Beta-Adrenergic receptor interactions. Direct comparison of receptor interaction and biological activity. Iodohydroxybenzylpindolol (I-HYP) is a chemically defined, high affinity, high specific activity beta-adrenergic antagonist that interacts with a single site on the turkey erythrocyte membrane. Study of the interaction of agonists, antagonists, and congeners with this site and concomitant alterations in adenylate cyclase activity have been carried out in the presence of high or low concentrations of guanine nucleotide. The results help clarify the relationship between binding and activation or inhibition of adenylate cyclase and the role of guanine nucleotides in modulating this interaction. There is a close correlation between binding constants (KD) for inhibitors determined by analysis of competitive displacement of 125I-HYP from receptor, and apparent affinities (Ki) for inhibition of adenylate cyclase. For activators, however, there is up to a 10-fold difference between KD and apparent affinity (KDapp) for adenylate cyclase activation at low guanine nucleotide concentration (10(-6) M guanylylimidodiphosphate). This difference is virtually abolished by employing higher nucleotide concentrations (10(-5) M guanylylimidodiphosphate) without significantly altering receptor affinity. This suggests that guanine nucleotides act by modulating receptor-enzyme interactions rather than hormone-receptor interactions. Moreover, several beta-adrenergic analogs previously shown to have no effect on adenylate cyclase in the absence of nucleotide, are partial agonists in the presence of 10(-5) M guanylylimidodiphosphate. Parallel analyses for a series of agonists and antagonists for adenylate cyclase activation and receptor interaction show affinities for levorotatory isomers generally 100-fold greater than for dextrorotatory isomers. Thus stereoconfiguration at the beta carbon clearly influences affinity of agonists or antagonists. Affinity is also importantly influenced by the nature of the aromatic ring as well as the N-alkyl group. The complexity of structure-function relationships for these compounds requires a redefinition of structural requirements for beta-adrenergic activity.


Quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase. (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. Cross-linking of purified NaK-ATPase with the (o-phenanthroline)2-cupric ion complex (CP) results in the reversible formation of dimers, trimers, tetramers, and pentamers of the large polypeptide and loss of NaK-ATPase activity. ATPase activity is partially recovered if NaK-ATPase is incubated with beta-mercaptoethanol after treatment with CP. In contrast to these results, if NaK-ATPase is cross-linked in crude canine kidney microsomes, only a dimer of the large polypeptide is formed. No cross-linking of the sialoglycoprotein to the large polypeptide is detected when NaK-ATPase is cross-linked in purified form. However, when NaK-ATPase is reacted with CP in either purified or microsomal form, the sialoglycoprotein cross-links to itself yielding a high molecular weight aggregate. The results show that the functional subunit structure of NaK-ATPase consists of at least two large polypeptides.


Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. The state of aggregation found in water-soluble preparations of an hepatic membrane protein responsible for the clearance of serum asialoglycoproteins has been shown to result from the self-associating properties of a single oligomeric protein. The smallest functional unit identifiable in aqueous solution possessed an estimated molecular weight of 500,000 with each of the successive components increasing in size by an equal amount to form an oligomeric series bearing an integral ratio of 1:2:3:4:5. The tendency towards self-association was promptly and completely reversed by the addition of Triton X-100 with the concomitant appearance of a single component. Extensive treatment with sodium dodecyl sulfate permitted the identification and isolation of two subunits with estimated molecular weights of 48,000 and 40,000, respectively. Amino acid and carbohydrate analyses revealed both subunits to be glycoproteins with a closely similar, but not identical, composition.


Phospholipids as ionophores.

Phospholipids as ionophores. The ionophoretic capabilities of phospholipids have been examined by direct measurement in a Pressman cell of the phospholipid-mediated translocation of cations across an organic phase separating two aqueous phases. Cardiolipin and phosphatidic acid were the most active inonophores among the phospholipids tested, with activities comparable to that of X537A in respect to the translocation of divalent cations. Cardiolipin translocates both divalent and monovalent cations at approximately equal rates. The ionophoretic activity of cardiolipin could be modulated by other phospholipids (inhibition), by butacaine (stimulation), by complexation with cytochrome c (inhibition), and by ruthenium red and lanthanum (inhibition). The rate of translocation of cations mediated by cardiolipin was independent of pH over a wide pH range (5.4 to 8.3). The same general pattern of properties observed for cardiolipin applied to phosphatidic acid except for stimulation by butacaine. Complexation of phospholipid mixtures, such as asolectin or mitochondrial lipid, with reduced cytochrome c, enhanced the ionophoretic capability of these phospholipids by 1 order of magnitude. The complex thus formed has the properties of a polyionophore. The possible physiological significance of this enormous ionophoretic potential of phospholipids is examined.


Ribonuclease S-peptide. A model for molecular recognition. The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.


Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine. Total protein synthesis in perfused livers of fed rats was determined by measuring the rate of valine incorporation based on the specific activity of valine attached to tRNA. Rates were not significantly altered when perfusate valine was increased from 0.40 to 5 mM and were similar to values calculated earlier from the specific activity of extracellular valine at a concentration of 15 mM. Overall protein degradation, computed from the sum of the rates of synthesis and the total increase of free intra- and extracellular valine, corresponded closely to the increase of free valine that occurred between 5 and 15 min after the addition of cycloheximide. In the latter experiments advantage was taken of the fact that the previously established suppressive effect of cycloheximide on proteolysis does not begin initially with the inhibition of synthesis, but 15 min later. Thus, the release of valine from 5 to 15 min was assumed to represent rates of protein degradation in effect prior to the addition of cycloheximide. The close agreement found among these independent assessments of protein metabolism thus appears to eliminate much of the previous uncertainty in the quantitation of hepatic protein turnover. In the course of these studies we noted that the specific activity of valyl-tRNA attained steady state values that were intermediate between specific activities of the extracellular and intracellular pools, but appeared to reach a steady state sooner than that of intracellular valine. To evaluate these early events more precisely, the specific activity of valine in tRNA and the intracellular pool was measured in a series of single-pass perfusion experiments where extracellular valine concentration and specific activity were held constant. The intracellular valine specific activity rose with a half-life of 1.2 min. By contrast, the rise in the specific activity of valyl-tRNA was biphasic: the initial phase of the valyl-tRNA curve was rapid, while the second phase had a half-life equal to that of intracellular valine. These data show that at physiological concentrations of valine, valyl-tRNA derives its amino acids from both the extracellular and cytoplasmic pools, and that at least some tRNA is charged by extracellular amino acids before they mix with intracellular amino acid pools, possibly from a precursor pool at or near the cell membrane.


Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline. The uptake and metabolism of [14C]choline in dissociated rat brain embryo cell cultures was examined as a function of the extracellular choline concentration. Choline uptake did not follow normal Michaelis-Menten kinetics, but rather exhibited two components with apparent Km of 0.016 mM and 0.96 mM. At low choline concentrations (high affinity uptake) most of the [14C]choline label was present in the phosphocholine fraction prior to the appearance of label in phospholipids. At high choline concentrations (low affinity uptake) a large proportion of the radioactivity was converted into acetylcholine. The dissimilarities between the formation of phosphocholine and acetylcholine as a function of choline concentration might be explained by the existence of two mutually independent enzymatic activities with different Km affinities for choline. Kinetic data augmented by double label studies, suggested that formation of choline phosphoglyceride proceeds entirely via a phosphocholine intermediate. Nearly all radioactivity in the lipid fraction is incorporated into choline phosphoglycerides. A higher turnover rate of choline incorporation into choline phosphoglycerides, accompanied by an increase in the levels of glycerophosphocholine, was observed in older cultures as compared to younger cultures. The metabolic implications of these findings in cultured brain cells in comparison with other in vitro systems are discussed.


Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A. In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.


On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles. The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.


Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide. In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.


Control of pyrimidine biosynthesis in human lymphocytes. Inhibitory effect of guanine and guanosine on induction of enzymes for pyrimidine biosynthesis de novo in phytohemagglutinin-stimulated lymphocytes. Human peripheral lymphocytes were incubated with Phaseolus vulgaris phytohemagglutinin. The induction of glutamine-utilizing carbamyl phosphate synthetase (EC 2.7.2.5) and aspartate transcarbamylase (EC 2.1.3.2) for pyrimidine biosynthesis de novo and the induction of uridine kinase were observed as described previously (Ito, K., and Uchino, H. (1971) J. Biol. Chem. 246, 4060-4065; Ito, K., and Uchino, H. (1973) J. Biol. Chem. 248, 389-392; Lucas, Z.J. (1967) Science 156, 1237-1240). By the addition of 1 mM guanine to the culture, the induction of the former two enzymes was inhibited, while that of uridine kinase was not, and even accelerated. An increase in the rate of [14C] bicarbonate incorporation into the acid-soluble uridine nucleotides via the de novo pathway for pyrimidine biosynthesis after phytohemagglutinin stimulation was inhibited by guanine, the incorporation rate being almost at the level of the control culture without phytohemagglutinin. Guanosine had a similar effect on pyrimidine biosynthesis. The induction of the three enzymes mentioned above was completely inhibited by adenine (1 mM). Guanine and guanosine seem to have a unique inhibitory effect on the induction of glutamine-utilizing carbamyl phosphate synthetase and aspartate transcarbamylase.


Assay of protamine messenger RNA from rainbow trout testis. A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.


Purification and properties of biologically active rainbow trout testis protamine mRNA. At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.


Purification of and mechanism studies on citrate synthase. Use of biospecific adsorption-elution techniques. Citrate synthases from animal tissues were found to bind to Sepharose-"ATP." A pure preparation of citrate synthase was obtained from a crude fraction of rat heart by the specific elution  of the enzyme from the Sepharose-"ATP" with the dead end complex-forming substrates, oxalacetate and CoA. The proposed mechanisms of citrate synthase, obtained from steady state kinetics, were examined in light of the elution pattern of the enzyme obtained using combinations of substrates and substrate analogs.


Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity. Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.


Identification and subcellular distribution of adipocyte peptides and phosphopeptides. Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.


Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain. The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.


Carbohydrate composition of bovine rhodopsin. The carbohydrate content of bovine rhodopsin was investigated and found to be different from previously reported values. Rod outer segments were isolated from dark-adapted bovine retinas by sucrose flotation and purified by sucrose density contrifugation. Rhodopsin was extracted with detergents and purified by chromatographic procedures involving calcium phosphate/celite chromatography followed by affinity chromatograpy on concanavalin A-Sepharose (or in some cases, gel filtration on agarose). Purified preparations of rhodopsin had A278/A498 ratios of 1.6 to 2.0. After treatment of the rhodopsin with chloroform/methanol (2/1) to remove lipids and detergents, the carbohydrate content was measured by gas-liquid chromatography, colorimetric and enzymatic analyses, paper chromatography, and electrophoresis. Rhodopsin was found to have about 9 mol of mannose and 5 mol of glucosamine per mol of visual pigment. A molar ratio of mannose/glucosamine of about 2 was also found in samples of rhodopsin obtained from two other laboratories. The amino acid analysis was similar to previously published values.


Inactivation of phosphorylase b by potassium ferrate, a new reactive analogue of the phosphate group. Rabbit muscle phosphorylase b reacts with the phosphate-like reagent potassium ferrate, K2FeO4, a potent oxidizing agent. The reaction results in inactivation of the enzyme and abolition of the ability of the enzyme to bind 5'-AMP. Activating and nonactivating nucleotides which bind at the 5'-AMP binding site such as 5'-AMP, 2'-AMP, 3'-AMP, and 5'-IMP substantially protect the enzyme from inactivation by ferrate. One to two residues of tyrosine and approximately 1 residue of cysteine are modified by ferrate under the conditions employed. Tyrosine is protected by 5-AMP, whereas cysteine is not. The tyrosine modification is suggested as the inactivating chemical reaction. The location of the inactivating reaction is suggested to be in or near the 5'-AMP binding site. The structural and chemical properties of ferrate ion are discussed and compared to those of phosphate. Ferrate ion may be a reagent useful for phosphate group binding site-directed modification of proteins.


Partial puridication of a membrane protein from human erythrocytes involved in glucose transport. In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.


Studies on the mechanism of 3-ketosphinganine synthetase. The biosynthesis of sphinganine and 4-D-hydroxysphinganine was studied in rat liver microsomes and whole cells of yeast (Hansenula ciferri). It was shown in both cases that the condensation of [2,3,3-2H3]serine and palmitic acid yielded long chain bases containing only two deuterium atoms, both of which were located on the terminal (C-1) carbon atom by combined gas-liquid chromatography/mass spectrometry. When the reaction with the liver microsomal system was carried out in 2H2O with the protium species of serine, the sphinganine contained a deuterium atom on C-2. These results suggest that the synthesis of 3-ketosphinganine involves the replacement of the alpha-hydrogen atom and the carboxyl group of serine by a proton from the medium and a palmitoyl group, rather than a previously proposed mechanism in which the alpha-hydrogen of serine is retained. Some stereochemical requirements of 3-ketosphinganine synthetase are discussed.


Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity. Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.


Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione. 3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.


In vivo metabolism of 4'-deoxypyridoxine in rat and man. In rats 80 to 95% of 4'-deoxypyridoxine administered intraperitoneally, intravenously, intramuscularly, or subcutaneously was excreted in the urine within 7.5 hours. Orally administered deoxypyridoxine was also rapidly eliminated. Over one-half of the excreted material appeared as deoxypyridoxine-3-(hydrogen sulfate) and the remainder as unchanged deoxypyridoxine. Tissue concentrations of deoxypyridoxine 5'phosphate were comparable to those of pyridoxal 5'phosphate. In normal men about 50% of a single oral dose (3 to 7.5 mg/kg of body weight) appeared in the urine within 6 hours. 4'Deoxy-5-pyridoxic acid accounted for 50 to 100% of the excreted material. The remainder was unchanged deoxypyridoxine. No deoxypyridoxine-3-(hydrogen sulfate) was detected in human urine and no 4'-deoxy-5-pyridoxic acid was found in rat urine. Deoxypyridoxine 5'-phosphate was not detected in the urine of either species. The complexity of deoxypyridoxine metabolism indicated by these data suggests the use of caution in extrapolating data obtained with deoxypyridoxine to B6 metabolism in the absence of deoxypyridoxine, and particularly in extrapolating results from the rat to man. Synthesis for 4'-deoxypyridoxine-3-(ethyl carbonate), 4'-deoxypyridoxine 5'-acetate, 4'-deoxy-3-0-(2-sulfoethyl)-pyridoxine, and the metabolites are presented. These synthesis were facilitated by using ethylchloroformate conjugates and N-methylpiperazine hydrolysis to block and unblock the phenol group.


Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid. Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.


The thrombin-like enzyme from Bothrops atrox snake venom. Properties of the enzyme purified by affinity chromatography on p-aminobenzamidine-substituted agarose. The trhombin-like activities from the snake venoms of two subspecies of Bothrops atrox, moojeni (type I) and marajoensis (type II), were purified to homogeneity by affinity chromatography on a support consisting of the inhibitor, p-aminobenzamidine, linked to Sepharose 4B with a spacer of diaminodipropylaminosuccinate. At room temperature the enzyme was not bound to the affinity support but rather was retarded in relation to the unbound protein. As a result the thrombin-like activity eluted in a large volume following the main protein fraction. However, at 4 degrees the enzyme was absorbed to the affinity support and could be eluted specifically with the ligand benzamidine (0.15 M). Optimal conditions for column loading and washing were 0.05 M Tris.HCl/0.4 M NaCl, pH 9.0 at 4 degrees. The type I enzyme isolated in this manner showed a single major band on pH 8.9 disc gel electrophoresis as well as two minor bands. Further purification by isoelectric focusing yielded one major and two minor components. All three protein fractions had identical thrombin-like activities and amino acid composition. The major band had a specific activity of 210 to 230 NIH thrombin units/mg, a S20, w of 2.65 S, a molecular weight of 29,000, and an E1% 280 of 15.6. This protein has a carbohydrate content, measured as hexose, glucosamine, and sialic acid, of 27%. From the amino acid and carbohydrate composition a partial specific volume of 0.700 ml/g was calculated. The type I enzyme, purified on affinity chromatography only, did not activate Factor XIII and was free of thromboplastin-like activity. The type II enzyme behaved very differently from the type I on pH 8.9 polyacrylamide disc gels yielding two major bands and two minor bands. The relative amounts of these four bands were not a function of purity. The type II enzyme had a specific activity of 650 to 700 NIH thrombin units/mg, a S20, w of 2.60, and a molecular weight of 31,400.


Allosteric transitions in cobalt hemoglobins. Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.


Derivatives of Clostridium acidi-urici ferredoxin containing altered amino acid sequences. Semisynthetic synthesis, biological activity, and stability. The semisynthetic syntheses and some properties of derivatives of Clostridium acidi-urici ferredoxin that contain amino acid deletions or replacements in the peptide chain are described. All 16 stable derivatives prepared, with the exception of [Trp2]ferredoxin, were fully active as electron carriers in the two enzymatic assay systems tested: the phosphoroclastic system and the ferrodoxin-dependent reduction of cytochrome c. E1Trp1]Ferredoxin had 70% of the activity of native ferredoxin in both assay systems. The stability in aerobic solution of [Ala1]ferredoxin, which had had its natural alanyl NH2-terminal residue removed and then replaced chemically, is the same as that of the native ferrodoxin (half-life of approximately 54 days). The relative stabilities of derivatives with a replacement or deletion of the NH2-terminal residue are as follows: [Ala1]- greater than or equal to [Phe1]-, [Lys1]-, [ Pro1]-, [Leu1]- greater than [Met1]- greater than [Gly1]- greater than [Glu1]- greater than des-Ala1-ferrodoxin. The data indicate that a large bulky residue, but not a  negatively charged residue, is tolerated in position 1 of the peptide chain and the greatly decreased stability (half-life = 1 day) of des-Ala1-ferredoxin confirms the importance of the NH2-terminal residue for the stability of the protein. The relative stabilities of derivatives containing Ala1, but including a replacement for the normal Tyr2, are as follows: Native greater than [Trp2]- greater than or equal to [Phe2]- greater than [His2]- greater than [Leu2]- greater than [Pro2]ferredoxin. [Gly2]- and des-Ala1-Tyr2-apoferredoxin did not form stable derivatives upon reconstitution with iron and sulfide, nor did [3-NO2-Tyr2, 30]- and [Leu2,3-NO2-Tyr30]apoferredoxins. Other relatively stable and fully active derivatives prepared included: [3-NH2-Tyr30]-, [3-F-Phe2]-, and [2-F-Phe2]ferredoxin. The behavior of these various derivatives demonstrates the importance of the peptide chain for the stability of C. acidi-urici ferredoxin and shows that the activity of ferredoxin can be altered by a single amino acid substitution in the peptide chain.


Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase. (a) The reactivity of substituted acetates as substrates for CoA transferase increases sharply with increasing basicity and exhibits a slope of 1.0 in a plot of either log kappacat or log (kappacat/Km) against pKa (betanuc = 1.0). This result shows that the catalyzed reaction, which involves both carboxylate activation and leaving group transfer, does not proceed through a fully concerted reaction mechanism in the rate-determining step. The result is consistent with a stepwise reaction mechanism that proceeds through an anhydride intermediate. (b) Equilibrium constants for thiol ester formation, either bound to the enzyme or free in solution, show the same dependence on the basicity of carboxylate ions (betaeq = 1.0) and are independent of acidity when expressed in terms of the carboxylic acid. Thus, the polar environment around substituents on the acyl group is the same for carboxylic acids, thiol esters, and oxygen esters. (c) The interaction of the terminal CH3CO group of acetoacetate with the active site causes a 200,000-fold increase in kappacat/Km, corresponding to a decrease in delta G++ OF 7.2 kcal/mol compared with an unsubstituted acid of the same pK. The binding energy of the coenzyme A moiety of the substrate is utilized to interact with the active site and cause a 10(4) to 10(6)-fold increase in kappacat, corresponding to a decrease in delta G++ of 6 to 9 kcal/mol, compared with fragments of the coenzyme A moiety added separatly or together. (d) The exchange of labeled coenzyme A into acyl-CoA substrates was found to be greater than or equal to 10(5) slower than substrate turnover.


Utilization of the inactivation rate of coenzyme A transferase by thiol reagents to determine properties of the enzyme-CoA intermediate. The rate of inactivation of succinyl-CoA:3-ketoacid coenzyme A transferase by thiol reagents is increased 3 to 100 times by very low concentrations of acyl-CoA substrates. The same maximum inactivation rate is found with acetoacetyl-CoA and succinyl-CoA. The enhanced rate of inactivation is caused by the stoichiometric formation of the enzyme-CoA intermediate and an accompanying conformation change of the enzyme. The inactivation rate provides a simple assay for the amount of enzyme present as the enzyme-CoA intermediate, using only catalytic concentrations of enzyme. This technique has been utilized to measure (a) a rate constant for hydrolysis of the enzyme-CoA intermediate of 0.10 min-1 at pH 8.1; (b) a stoichiometry of two active sites per enzyme molecule; and (c) the equilibrium constants for formation of the enzyme-CoA intermediate from dilute solutions of substrates (and hence for the overall reaction) by determining the ratio of [enzyme-CoA]/[enzyme] in the presence of a series of substrate "buffers" at different ratios of [RCOO-]/[RCOSCoA]. As the total concentration of acyl-CoA and carbosylate substrates is increased, the inactivation rate is decreased. This indicates that the Michaelis complexes are protected against inactivation.


Properties of biologically active messenger RNA from human placenta. Cell-free synthesis of two immunoreactive forms of placental lactogen. In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.


Group fractionation of eukaryotic ribosomal proteins. The proteins of the subunits of rat liver ribosomes were fractionated by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. The 40 S ribosomal proteins were separated into five groups containing between 3 and 14 proteins; the 60 S proteins, into seven groups of 3 to 15. Only a comparatively small number of proteins occurred in appreciable amounts in more than one group. The number of relatively acidic proteins associated with the ribosomal subunits was larger than had been reported before: it is not known if they are initiation or translation factors or ribosomal structural proteins. The group fractionation procedure has proven valuable as the initial step in the isolation and characterization of rat liver ribosomal proteins.


Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with beta-naphthoflavone or phenobarbital. The administration of polycyclic aromatic compounds such as beta-naphthoflavone or 3-methylcholanthrene is known to cause the induction of many liver microsomal monoxygenase activities and the appearance of a distinct cytochrome called P-448 in genetically responsive, but not in nonresponsive, inbred mouse strains. However, the administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin induces these activities and cytochrome P-448 formation to the same extent in both responsive and nonresponsive inbred strains. In contrast, phenobarbital or pregnenolone-16 alpha-carbonitrile induces in both responsive and nonresponsive strains a different profile of enzyme activities and the appearance of cytochrome P-450 (rather than cytochrome P-448). In the present studies, electrophoresis of liver microsomal proteins from inbred C57BL/6N and DBA/2N and recombinant inbred AKXL-38 and AKXL-38A mouse strains revealed the presence of four polypeptides whose relative staining intensity could be correlated with the induction state of the microsomes as determined by enzymatic and spectral methods. Of these four bands, Band 4 (55,000 daltons) was increased whenever spectral measurements revealed an increase in the cytochrome P-448 content due to administration of beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Administration of pregnenolone-16alpha-carbonitrile caused an increase in Band 3 (54,000 daltons), whereas administration of phenobarbital caused an increase primarily in Band 2 (51,000 daltons) but also smaller increases in Band 1 (49,000 daltons) and Band 4. The changes observed for phenobarbital and pregnenolone-16alpha-carbonitrile were the same for both responsive and nonresponsive strains. The same electrophoretic technique was used to measure the incorporation of radioactive leucine into microsomal proteins. Microsomes were prepared from liver combined from responsive mice (C57BL/6N) treated with beta-naphthoflavone and L-[14C]leucine and nonresponsive mice (DBA/2N) treated with beta-naphthoflavone and L-[3H-4,5]leucine. A significant increase in the 14C/3H ratio was observed for Band 4, and decreases were seen for Bands 1 and 2. In similar experiments with other mice and phenobarbital as the inducing agent with L-[14C]leucine and the vehicle alone with L-[3H-4,5]leucine, the 14C/3H ratio was markedly increased for Band 2, and smaller increases were observed for Bands 1 and 4. These results and other data presented indicate that the increased formation of cytochrome P-448 and P-450 by beta-naphthoflavone and phenobarbital, respectively, is primarily the result of an increased rate of de novo protein synthesis rather than a decreased degradation rate or a conversion of pre-existing polypeptides.


Purification and properties of a new polypeptide chain elongation factor, EF-1beta, from pig liver. Eukaryotic polypeptide elongation factor 1 (EF-1) from pig liver has been resolved into two complementary factors, EF-1alpha and EF-1beta (Iwasaki, K., Mizumoto, K., Tanka, M., and Kaziro, Y. (1973) J. Biochem. (Tokyo) 74, 849). This paper describes the procedures for purification of EF-1beta and some properties of the purified factor. The purification method includes an aqueous two-phase separation technique, a treatment of the crude factor with sodium cholate and two successive column chromatographies on diethyl-aminoethyl-Sephadex A-50. By this method, EF-1beta was purified about 50-fold starting from the material obtained after two-phase separation followed by ammonium sulfate fractionation with a recovery of 20%. The purified EF-1beta appeared homogeneous, having a molecular weight of about 90,000. It consisted of two unequal subunits of the molecular weights of 55,000 and 30,000. It stimulates polymerization of phenylalanine dependent on poly(U) in the presence of both EF-1alpha and EF-2, as well as the EF-1alpha-dependent binding of phenylalanyl-tRNA to ribosomes in the presence of GTP. However, it had no effect on the stoichiometric binding of phenylalanyl-tRNA to ribosomes dependent on EF-1alpha in the presence of guanyl-5'-yl methylenediphosphonate. These results indicate that the function of EF-1beta is to stimulate the recycling of EF-1alpha.


The influence of surface chemistry on implant interface histology: a theoretical basis for implant materials selection. A theory is proposed stating that an ideal implant material must have a dynamic surface chemistry that induces histological changes at the implant interface which would normally occur if the implant were not present. Evidence for the validity of this theory is provided with a series of bone-implant studies which result in stable interfacial osteogenesis under specific implant surface chemistry conditions. Insufficient or excess surface ion concentrations produce negative osteogenesis and fixation results. Implantation of osteogenic implants in soft tissues also produces undesirable histological responses as proposed in the theory. A variety of surface chemical analyses of the implant are reviewed which provide a scientific basis for the implant surface theory.


Biocompatibility of materials for total joint replacement. The clinical use of total joint prostheses demands absolute biocompatibility of the materials employed. The purpose of this experiment was the bioassay of some materials considered as possible candidates for use in total joint prostheses as load-bearing members or as wear-resistant surfaces. Some materials already in use were also tested. 316L stainless steel was used as a control. The materials were implanted as a standardized rod and in particulate form. An average of 12 samples per material were implanted in soft tissue for six months and a total of 145 rabbits were used in this study. Twenty-five materials including metals, polymers, and ceramics were tested in solid and powdered form. A semiquantitative evaluation of local tissue reaction and a study of organs was performed. Polymers and metallic materials showed in general a mild tissue reaction. Ceramics, which some authors describe as the best tolerated materials, elicited variable tissue responses. Some of these (glass-ceramics) presented very poor tissue tolerance. The least reactive, titanium oxide, titanium aluminate, and aluminum oxide, presented a degree of tissue reaction comparable to that of corrosion resistant metals, but not superior to them. Moderate reactivity was the general rule for particulate materials except for the Pyroceram glass-ceramics, polymides, and Teflon. Ultrahigh molecular weight polyethylene in particulate form elicited a rather cellular tissue response, a fact to be considered when projecting long-term results in total joint arthroplasty. No pathological changes compatible with systemic toxicity by materials tested were observed in the study of the organs.


Electrical stimulation of bone growth into porous A12O3. Porous, cylindrical, high alumina, ceramic structures were fabricated for implantation in the superior third of canine femoral medullary canals. Implant durations of one, two, four, and eight weeks using three animals per time period were studied. A constant current (10 muA) power supply was used to attempt to stimulate bone growth into the porous ceramic structures. Osseous tissue in-growth was evaluated using a mechanical push-out test, microadiography, and histological thin sectioning. The interfacial shear strength and tissue micro-structure was studied as a function of implant residence site and implant residence site and implant residence time. The results indicate that initially the proximal implants have a higher interfacial shear strength, however, after eight weeks of implantation the distal implants have a shear strength about twice that of the proximal implants. This trend is observed for both the electrically stimulated and nonstimulated specimens. The 10 muA current level used in this investigation was found to have a small but significant effect on shear strength increasing it at implantation times of less than eight weeks.


An in vitro study of bacterial response to inert and reactive metals and to methyl methacrylate. The clinical use of inert materials for internal fixation and joint replacement devices is increasing rapidly; studies on the effect of these materials on bacteria and host resistance to infection have not kept apace. Any substance placed within the body may interfere with host-parasite interaction, either by its physical presence or by physiochemical activity at its interface with the surrounding tissue. Either mechanism, by altering the normal host defense mechanism, may promote or retard bacterial growth. Bacterial growth may also be altered if a trace element essential to or inhibitory to the bacteria or an element that antagonizes or potentiates humoral antibacterial systems is leached from the substance. We have tested bacterial growth and inhibition in vitro in the presence of substances used as implant materials: surgical silver, iron, zinc-coated galvanized iron, aluminum alloy, stainless steel, Vitallium, and methyl methacrylate. Our results showed: 1) metals that have low grades of tissue reactivity and little bacteriotoxic effect provided a framework along which bacterial growth and propagation occurred; 2) metals that have higher levels of cellular reactivity, specifically aluminum and galvanized wire, caused selective bacterial toxicity. The in vitro response of bacteria to methyl methacrylate was similar to that of Vitallium and other inert substances, i.e., growth and propagation were abundant adjacent to methyl methacrylate. These studies led us to speculate that, if similar phenomena occur in vivo, bacterial growth and dissemination might be increased when an inert implant material was used and decreased when the implant material was more reactive.


Hyaluronidase-bound membrane as a biomaterial for implantable fuel cells. A new biomaterial containing covalently bound hyaluronidase was prepared. An application of this enzyme membrane is to improve the performance of an implantable fuel cell. Hyaluronic acid is a contributor to the viscosity of tissue fluids but can be a potential fuel source because of its sugar content. The incorporation of immobilized hyaluronidase would not only contribute to a more available fuel supply by splitting hyaluronic acid but, perhaps more importantly, enhance the rate of mass transport of  fuel, O2, and reaction products by reducing the viscosity near the electrode membranes. Hyaluronidase was bound to Sepharose gel and its thermoplastic membrane after activation by cyanogen bromide. Fourteen and 22% of the activities were recovered from the gel and membrane, respectively. The activity of the bound enzyme was stable for six months at 0 degrees C. The addition of hyaluronic acid, 1 mg/ml, to a typical implantable type bioautofuel cell in vitro increased external solution viscosity from 1.1 to 2.5-2.8 cP and reduced voltage output under 10 komega by 60% in 3 hr. When the hyaluronidase bound membrane was placed at the anode, viscosity of the glucose-hyaluronic acid solution was lowered to 1.8 cP and the cell output increased to the original level of a glucose-fueled cell in 3 hr. Glucosamine-equivalent released from hyaluronic acid at the electrode was 3.1 mg after 22.5 hr. This represents 90% of the theoretical consumption. Restoration of the cell output was probably a combination of the enhanced transport of fuel, O2 and products, and/or appearance of a new fuel, glucosamine-equivalent.


The rate of bone ingrowth into porous metal. Experiments have been devised to study the rate of ingrowth of bone into porous metal with pore sizes up to 100 mu and to study the significance of a gap between the porous metal surface and bone. When the porous coat was in direct apposition with bone, the implant was firmly locked in place after a three week period and the plateau value of implant-tissue shear strength was reached at four weeks. A gap of 1.5 mm between the bone and the implant was bridged by new bone within four weeks.


An evaluation of bone growth into porous high density polyethylene. The purpose of this investigation was to study bone growth into porous polyethylene rods as a function of time and pore structure. Previous studies have indicated the biocompatibility of solid polyethylene materials which are currently being used clinically. Porous polyethylene rods were implanted in the femurs of mongrel dogs which are sacrificed four, eight, and 16 weeks postoperatively. The implants were then sectioned and examined histologically and microadiographically. Quantitative techniques were employed to determine the amount of bone ingrowth as a function of time and pore size. The pore structures of the materials were evaluated using optical microscopy and mercury intrusion porosimetry. The results of this investigation have demonstrated that porous polyethylene is capable of accepting bone growth into pores as small as 40 mum. The optimum rate of bone ingrowth was observed in pore sizes of approximately 100 to 135 mum, with no increase in the rate of bone ingrowth observed in samples possessing larger pore sizes. No adverse tissue response was found at implant times up to 16 weeks in pore sizes of 100 mum or larger.


Classification of knee ligament instabilities. Part I. The medial compartment and cruciate ligaments. Based on the clinical and operative findings in sixty-eight knees with acute tears of the medial compartment and cruciate ligaments, a standardized terminology and classification of knee ligament instability is presented. With an intact posterior cruciate ligament, anteromedial, anterolateral, or posterolateral rotatory instability may occur, but not true posteromedial rotatory instability. With the posterior cruciate ligament ruptured, straight anterior, posterior, medial, or lateral instability may be found.


Classification of knee ligament instabilities. Part II. The lateral compartment. Lateral instability of the knee is less frequent but more disabling than medial instability of a comparable amount. At the same time the diagnostic tests for lateral instability are more subtle and more frequently misinterpreted. Posterolateral rotatory subluxation is demonstrated by an apparently positive posterior drawer test with the tibia in neutral rotation or by the external rotation-recurvatum test with the knee in extension. Anterolateral rotatory subluxation is present when the anterior drawer test with the tibia in neutral rotation demonstrates that the lateral tibial condyle appears to become more prominent or that both condyles appear to become equally prominent.


The anterior cruciate ligament. A functional analysis based on postmortem studies. In forty fresh human cadaver knees the function of the anterior cruciate ligament and of its two component parts, the posterolateral part and the anteromedial band, were studied by cutting these ligaments and others in different sequences and combinations and then manually stressing the knees. The anterior drawer sign cannot be obtained unless the anteromedial band is severed. The postolateral part and the medial collateral ligament are, respectively, the secondary and tertiary restraints limiting the anterior drawer sign. Both internal and external rotation are limited by the anterior cruciate ligament, especially when the knee is in extension. The anterior cruciate ligament also limits hyperextension.


Experimental stabilization of segmental defects in the human femur. A torsional study. One of each of thirty-five pairs of fresh intact femora were tested to failure in torsion, recording the dynamic torque, the absorbed energy, and the angle of rotation. These results were compared with the results obtained with the contralateral femur, reconstituted after removal of a segment. Intramedullary nails with polymethylmethacrylate cement, strips of titanium mesh with cement, bone plates with and without cement, and multiple Steinmann pins with cement were the reconstituting configurations. Bone plates were the strongest configuration; the failure torques in all cases were limited by the stress concentration effects of the holes in the bone used for screw fixation. The use of cement as an adjunct to single-plate fixation provided some additional strength. The torsional strength of femora fixed with Küntscher and Schneider nails was limited by failure of the cement and bone. The use of titanium mesh with polymethylmethacrylate was less effective, because this composite has a low torsional rigidity. The use of multiple Steinmann pins packed with polymethylmethacrylate in the medullary cavity should be discouraged because severe twisting and fragmentation of the surrounding acrylic will occur at low levels of torque.


A comparison of cortical bone atrophy secondary to fixation with plates with large differences in bending stiffness. Two internal-fixation plates with large differences in bending stiffness were applied to pairs of intact femora of six adult mongrel dogs to study the osteoporosis induced by rigid fixation. After nine and twelve months of plate application a significant, increased amount of bone atrophy was seen on the rigidly plated side. Biomechanical measurements of specimens from various segments of both femoral diaphyses showed the bones to have similar mechanical properties (as tissue), but different structural properties (as organ). These findings suggest that the osteoporosis due to rigid plate fixation occurs by thinning of the cortex rather than by reduction of the mechanical properties of the osseous tissue.


Normal and abnormal motion of the shoulder. The roentgenographic parameters of motion in normal and abnormal shoulders, including the movement of the scapula, arm angle, glenohumeral angle, scapulothoracic angle, excursion of the humeral head, and instant center of motion for abduction in the plane of the scapula, were determined in twelve normal subjects and fifteen patients. The scapula rotated externally with abduction. The ratio of glenohumeral to scapulothoracic movement was 5:4 after about 30 degrees of abduction. The center of rotation of the glenohumeral joint for abduction in the plane of the scapula was located within six millimeters of the geometric center of the humeral ball. The average excursion of the humeral ball on the face of the glenoid in the superoinferior plane between each 30-degree arc of motion was less than 1.5 millimeters in normal subjects. Significant previous injury resulting in abnormal mechanics of the shoulder joint was associated with abnormal values for excursion of the instant center and of the humeral head. An abnormal glenohumeral-to-scapulothoracic ratio was associated with significant pain in the shoulder. The fact that these various parameters were sensitive indicators of normal and abnormal motion raises the possibility of diagnostic clinical application.


Electromyography before and after surgery for hip deformity in children with cerebral palsy. A comparison of clinical and electromyographic findings. Twenty-three ambulatory children with spastic diplegic cerebral palsy were evaluated clinically and by electromyography before and after hip-muscle surgery. The stretch tests originally designed to distinguish specific muscle tightness and spasticity were found to be non-specific when tested by electromyography. Ambulatory electromyograms using needle electrodes and telemetry generally showed decreased activity in the released muscles and, on occasion, changes in activity in muscles not operated on. These unanticipated changes after release may explain some of the unpredictability of results of such procedures in cerebral palsy.


Femoral trabecular-pattern index and bone mineral content measurement by photon absorption in senile osteoporosis. A comparative study of the usefulness of the femoral trabecular-pattern index (Singh index) and bone mineral content measurement by photon absorption in senile osteoporosis was performed in 106 white women, seventy to ninety-five years old. In 17% of the subjects adequate hip roentgenograms could not be obtained. Interpretation of the Singh index was not found to be simple and easily reproducible. A positive correlation was noted between the bone mineral content and the Singh index. The incidence of previous vertebral and non-vertebral fracture was noted to be inversely related to both bone mineral content and the Singh index, but neither method clearly separated subjects with fracture from those without fracture.


The fate of hypertrophic chondrocytes of the epiphyseal plate. An electron microscopic study. An ultrastructural study of the distal femoral epiphyseal-metaphyseal junction of C3H mice revealed that the hypertrophic chondrocytes undergo degenerative changes leading to their death. While most chondrocytes will die before their lacunae are opened to the marrow, others survive for a time in lacunae which communicate with the primary spongiosa, but they also ultimately die. There was no evidence to indicate that hypertrophic chondrocytes survive or are transformed into other cells.


Total hip replacement in patients with ankylosing spondylitis with involvement of the hip. Twenty-three patients with ankylosing spondylitis underwent thirty-four total hip arthroplasties with an average follow-up of 42.5 months. The over-all results were rated good in all but two patients. Significant benefit was obtained in posture and function so that these patients could be gainfully employed. Improvement in the sum total range of motion was 96.1 degrees in patients with bone ankylosis and 150 degrees in those with fibrous ankylosis. The preoperative sum total range of motion was maintained or improved in hips with a painful arc of motion. Most of the limited motion in patients with ankylosed hips was due to myositis ossificans.


Tibial lengthening using a new extension device. Report of thirty-two cases. Using a new type of external fixation device for lengthening the tibia, lengthenings of from 3.5 to 6.5 centimeters were accomplished in thirty-two children, eight to seventeen years old, the six complications, none of which jeopardized the result. The procedure was painless and the patients were ambulatory on crutches two to three days after operation, while lengthening was continued as an out-patient procedure.


Batchelor's extra-articular subtalar arthrodesis. A report on sixty-four procedures in patients with poliomyelitic deformities. Sixty-four feet of fifty-four patients treated with Batchelor's extra-articular subtalar arthrodesis for poliomyelitic deformities were reviewed after a minimum follow-up of four years. Early in the series the results were poor because of inexperience with the technique and poor selection of patients. The incidence of non-union, which appeared to be high in this series compared with other series in which the Grice technique was used, was attributed mainly to the more advanced age of our patients at the time of arthrodesis (average age, 9.9 years). A common factor in the cause of non-union was tightness of the heel cord. Redistribution of muscles to balance power in the foot did not appear to be necessary to achieve good results in feet with equinovalgus deformity, and conversely, energetic redistribution of muscle power in these feet did not result in late varus deformity.


Bucket-handle tear of the medial meniscus. A case for conservative surgery. To compare the results of two methods of treatment for bucket-handle tears of the medial meniscus eighty-seven knees were studied, thirty-seven treated by excision of the bucket-handle tear alone and fifty by total meniscectomy. A postoperative history suggestive of locking was given by one patient in each treatment group. Anteroposterior laxity was found in 72% of knees treated by excision of the bucket-handle tear alone as compared with 68% of the knees treated by total meniscectomy, but in all other respects the results showed a marginal preference in favor of the simpler operation. Although the numbers are too small to prove that results are better following excision of the bucket-handle tear alone, it is suggested, in the absence of evidence that total meniscectomy is more advantageous, that the simpler operation of excision of just the bucket-handle tear is the treatment of choice in bucket-handle tears of the medial meniscus.


Recurrent anterior dislocation of the shoulder. Long-term follow-up of the Putti-Platt and Bankart procedures. Follow-up of 176 patients among 223 who underwent 232 repairs for recurrent shoulder dislocation (average follow-up, 10.2 years) revealed a recurrence rate of 11%. Among the twenty with recurrence, important factors were youth, athletic activity, inadequate immobilization, a history of contralateral dislocation, and a family history of shoulder dislocation. Redislocation occurred two years or more after operation in seven of the twenty patients with recurrence. Surgical repair permitted good function without significant pain in most cases.


The modified Bristow procedure for recurrent dislocation of the shoulder. A review of fifty-one cases of the modified Bristow procedure for recurrent anterior shoulder instability is presented. The results were favorable. The redislocation rate was 2% with few complications. The average limitation of motion was 11 degrees of external rotation. Athletic individuals with involvement of the dominant shoulder were not capable of returning to high performance levels of overhead sports activity (particularly throwing) after the operation.


Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains. Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freeze-fracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles-trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (76 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18 degrees C), while in cells grown at the nonpermissive temperature (27 degrees C) the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses", also found in wild-type and in nd9 (18 degrees C) cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.


Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. The distribution and quantitation of 125I-alpha-bungarotoxin (alpha-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, alpha-BTX was bound to approximately 90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of alpha-BTX relative to this membrane was determined to be 46,000 +/- 27% sites/mum2.


Involvement of vesicle coat material in casein secretion and surface regeneration. The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.


Structural complexes in the squid giant axon membrane sensitive to ionic concentrations and cardiac glycosides. Giant nerve fibers of squid Sepioteuthis sepioidea were incubated for 10 min in artificial sea water (ASW) under control conditions, in the absence of various ions, and in the presence of cardiac glycosides. The nerve fibers were fixed in OsO4 and embedded in Epon, and structural complexes along the axolemma were studied.


Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate. Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.


Carbamycholine prevents giant granule-formation in cultured fibroblasts from beige (Chediak-Higashi) mice. Primary embryonic fibroblasts isolated from beige (Chediak-Higashi) mice develop pathognomonic giant granules in vitro. Inclusion in the culture medium of carbamylcholine (carbachol) or phorbol myristate acetate (PMA) results in the generation of morphologically normally granules. Chediak-Higashi fibroblasts are highly susceptible to shape changes induced by colchicine. This abnormal property is also corrected by carbachol and PMA.


Intranuclear complexes in a copper-tolerant green alga. The results of ultrastructural studies and transmission electron microscope microanalysis of two Scenedesmus strains experimentally exposed to copper sulfate are presented. A fine-structural examination of the cells revealed the presence of nuclear inclusions in the form of central dense-core complexes. Cytoplasmic structures resembling the intranuclear inclusions were occasionally found in the cells. TEM-X-ray microanalysis of these structures has provided evidence that the inclusions contain copper. It is concluded that their presence may be regarded as a detoxifying mechanism.


Cautionary note on the use of [methyl-3H]thymidine to measure rates of chloroplast DNA synthesis in Chlamydomonas reinhardtii. Commercial [methyl-3H]thymidine preparations tested here contain about a 0.2% contaminant which is rapidly incorporated into Chlamydomonas DNA. This contaminant obscures the measurement of the rate of chloroplast DNA synthesis when methyl-labeled preparations are used. Such contaminants are not present in ring-labeled (either 3H or 14C) thymidine preparations. In ring-labeled thymidine preparations, a slower incorporation rate commensurate with cell density is observed. These slower, long-term incorporation kinetics would be expected for the utilization of bona fide thymidine into chloroplast DNA.


Oscillations of calcium ion concentrations in Physarum polycephalum. Aequorin is a photoprotein which emits light in response to changes in free calcium concentration. When aequorin was microinjected into plasmodia of Physarum polycephalum, light emission varied in synchrony with the motile oscillations of the organisms. Therefore, movement is correlated which changes in the concentration of free calcium.


Rhodopsin in the rod outer segment plasma membrane. Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.


Relationship between chromosome condensation and metaphase lysine-rich histone phosphorylation. Treatment of metaphase HTC cells with ZnCl2 inhibits histone phosphatase activity and leads to an increase in the hyperphosphorylated forms of the lysine-rich (F1) histone. Under normal conditions a massive phosphatase activity is triggered as the cells shift from M into G1 phase. In the presence of ZnCl2 this activity is abolished and thehyperphosphorylated form of F1 persists intact into G1. We have asked the simple question of whether the chromosome can still extend during the M-G1 transition even if the F1 histone is maintained in the hyperphosphorylated form. We observe an apparently normal extension os the chromosomal material under these conditions, though it is evident that high levels of ZnCl2 have rather substantial effects on other cell functions.


The polymerization of actin. II. How nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes. At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage, in the accumulation of actin in the periacrosomal region is the actin sequested in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrsomoal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state.


Morphological changes in tight junctions of Necturus maculosus proximal tubules undergoing saline diuresis. Tight junctions between epithelial cells are believed to control the paracellular diffusion of substances across epithelia. Epithelia in which tight junctions are poorly developed display a higher paracellular electrical conductance, while those with extensive tight junctions show lower conductance values. We described here a particular epithelium, that of the proximal tubules of the Necturus kidney, in which the development of the tight junctions varies in parallel with a change of paracellular electrical conductance. In control conditions, tight junctions between epithelial cells of the proximal tubules are more developed than in tubules undergoing saline diuresis, a situation which increases the conductance across the paracellular shunt pathway.


Non-histone protein synthesis during G1 phase and its relation to DNA replication. The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.


Effect of exogenous ATP on sodium transport in mammalian red cells. The effect of exogenous adenosine triphosphate (ATP) and other nucleotides on the transport of Na in various mammalian red cells has been studied. While they have no effect on the transport of Na in human and cat red cells, in dog red cells adenosine and its mono-, di- and triphosphorylated forms were found to increase Na-influx. Of these, ATP has the most striking effect, causing a more than 8-fold increase at a concentration of 0.6 mM and exerting this effect at a dose range of 10(-5) to 10(-3) M. The effect of ATP is rapid (less than 5 minutes) and can be reversed by washing or the addition of calcium or magnesium. In contrast to the adenosine series other phosphorylated nucleotides (GTP, CTP, UDP, GDP and cAMP) have no effect. The well known volume dependent Na-transport in these cells is reversed in the presence of 0.6 mM ATP. It is suggested that ATP acts on passive cation movements either by chelation of membrane charge or by a direct interaction with  membrane proteins and may be involved in the volume regulation of cation transport in the dog erythrocyte.


Methionine metabolism in BHK cells: selection and characterization of ethionine resistant clones. The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo-B12. Clones resistant to 30 mug/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 X 10(-5). An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone. Measurement of the specific activity of menadione reductase, B12 methyltransferase and ATP: L-methionine S-adenosyl-transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback-inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase. These results suggest that the pathway of re-use of S-adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.


Bromodeoxyuridine induced variations in the level of alkaline phosphatase in several human heteroploid cell lines. The level of alkaline phosphatase in a number of established cell lines of human origin can be modified by exposure to non-lethal concentrations of bromodeoxyuridine (BRdU). In the several cell lines examined an inverse relationship between amount of induction and constitutive level of the enzyme was observed. Thus, the H.Ep 2 line, which had the highest basal level of enzyme, was reversibly repressed following exposure to the drug, whereas other cell lines with relatively low constitutive enzyme levels were induced to a maximum of 10-fold following exposure. Initiation of induction required from 24 to 48 hours, and as short an exposure ("pulse") as five hours was sufficient to produce induction. Exposure to visible light had no effect upon the repression of alkaline phosphatase in H.Ep 2 by BRdU. Induction did not occur in non-dividing, serum starved cells. The time course of induction by BRdU and hydrocortisone was similar, and simultaneous exposure  of the cells to both agents resulted in no greater induction than that observed with either drug alone. Experiments utilizing mitomycin C yielded significant induction in the presence of this agent alone, and somewhat less induction when both mitomycin C and BRdU were added simultaneously. These results suggest that DNA synthesis is required for BRdU were added simultaneously. These results suggest that DNA synthesis is required for BRdU-mediated induction of alkaline phosphatase.


Studies on steroids. CIX. O-omega-haloalkyloximes, new derivatives for gas chromatography-mass spectrometry of oxosteroids. Several kinds of omega-haloalkoxyamines have been prepared as new derivative-forming agents applicable for the gas chromatography-mass spectrometry of carbonyl compounds. Of these reagents, 2-chloroethoxyamine hydrochloride [O-(2-chloroethyl)hydroxylamine] was the most suitable for use with oxosteroids because of its higher reactivity and the appropriate retention value and satisfactory chromatographic properties of the resulting oximes. The contribution to the retention value due to formation of the 2-chloroethyloxime was determined for 5alpha-androstanones having the oxo-group at various positions in the steroid nucleus. As expected, 17-oxosteroid O-2-haloethyloximes exhibit readily distinguishable isotope peaks as a cluster in the mas spectrum.


Separation of C19 and C21 dihydroxysteroids by open-hole tubular glass columns and lipophilic gel chromatography. The combination of lipophilic gel chromatography and high-resolution glass capillary chromatography has been shown to be an important means of identification and quantification of steroid urinary metabolites. Some problems in the use of capillary columns, such as coating with polar and apolar stationary phases, column performance, thermal stability and injection systems have been investigated and this type of chromatography has been used in the analysis of 33 dihydroxysteroids. Gel chromatography is a method complementary to high-efficiency gas chromatography. The steroids are eluted according to the number of hydroxyl groups in the molecules.


Studies on monoterpene glucosides and related natural products. XXXI. Gas chromatography and gas chromatography-mass spectrometry of iridoid and secoiridoid glucosides. A total of 33 iridoid and secoiridoid glucosides were detected by gas chromatography on several columns such as OV-1 or OV-17. Representative glucosides were then subjected to gas chromatography-mass spectrometry, giving some characteristic peaks that permitted the discrimination of both types of glucosides from other compounds in most instances. The successful detection of both types of glucosides in several plant extracts showed the applicability of this combination of methods to small amounts of plant materials.


Quantitative thin-layer chromatographic analysis of ergotamine tartrate and caffeine in the nanogram range. Direct, quantitative, thin-layer chromatographic methods for the determination of ergotamine tartrate and caffeine in the presence of each other in blood and in pharmaceutical preparations are described. The blood is centrifuged, the plasma decanted from the coagulum and deproteinized with acetone-methanol. After removal of the solvent mixture, the active ingredients are extracted from the remaining aqueous solution with chloroform. In the case of the pharmaceutical preparations, the active ingredients are also extracted with chloroform or methanol. Ergotamine tartrate and caffeine are separated on pre-coated silica gel 60 F254 plates and measured directly on the thin-layer plate, ergotamine tartrate being determined by the fluorescence method, excitation wavelength 365 nm, at lambdamax.=450 nm and caffeine by the reflactance method at lambdamax.=274 nm. The analytical methods are suitable for the bioavailability studies of these drugs in blood as they enable the determination of 20 ng ergotamine tartrate with a coefficient of variation of 6.7% and the determination of 200 ng caffeine with a coefficient of variation of 6.1%. The methods have also proved useful in the determination of the active ingredients of drug forms such as tablets and suppositories and can be well reproduced with a maximum coefficient of variation of 4.9% for ergotamine tartrate and 3.5% for caffeine.


Quantitative determination of naltrexone and beta-naltrexol in human plasma using electron detection. A gas-liquid chromatographic method is described for the determination of naltrexone and beta-naltrexol in human plasma following derivatization with pentafluoropropionic anhydride using electron capture detection. The lower sensitivity of the method for absolute standards is 5-10 pg. Following an acute 100-mg dose to a subject, peak levels of naltrexone of 15 ng/ml at 2 h and of beta-naltrexol 84 ng/ml at 4 h were observed. The levels of both compounds decreased by 24 h after the dose: naltrexone to 2.9 ng/ml and beta-naltrexol to 25 ng/ml. Following chronic administration for two weeks of 100 mg per day the peak levels of naltrexone and betanaltrexol increased to 26.9 and 131 ng/ml at 2 h, respectively, but by 24 h both compounds were at levels similar to those following a single dose. Thus no accumulation of either drug ro metabolite in the plasma was seen following chronic naltrexone administration.