Immune-complex involvement in the pathogenesis of infectious bursal disease virus in chickens. Frozen kidney sections from chickens inoculated with infectious bursal disease virus (IBDV) were stained with fluorescein-conjugated rabbit anti-chicken gamma-globulin. Fluorescence was observed in the renal glomeruli of infected chickens, indicating that gamma-globulins, probably in the form of immune complexes, had lodged in the glomeruli of IBDV-infected chickens. This suggests that immune complexes may play an important role in the pathogenesis of IBDV infections in chickens.


Vigilance performance of men sleeping under arctic conditions. This study investigated the effect of cold exposure on vigilance performance in men working and sleeping under Arctic conditions. The results were compared with changes in the amount of REM sleep. EEG sleep recordings were made on six subjects during five baseline nights in laboratory conditions, 16 experimental nights in Arctic conditions, and four recovery nights in the laboratory. Vigilance tests were administered every second day during the experimental period and two times during each of the baseline and recovery periods. During the first night in the Arctic, the amount of REM sleep fell to 50% of baseline and a large decrement occurred in detection performance on the following morning. During the remaining nights in the cold, REM deprivation averaged about 25% with somewhat greater deprivation occurring during colder nights. Reaction time measures generally increased throughout the experimental period and subsequently showed incomplete recovery. However, detection performance gradually improved during the experimental period but showed some regression following colder-than-usual nights, when REM deprivation increased. This suggested that performance on this type of task may be related to temperature variations and changes in REM deprivation.


Complement-dependent antibody cytotoxicity test of chicken antibody with duck complement used against cells of a Marek's disease lymphoma-derived cell line (MSB-1). Complement-dependent antibody cytotoxicity (CDAC) against cells of a lymphoblastoid cell line (MSB-1) derived from Marek's disease lymphoma was investigated in a chicken antibody system using complements from several animal species. Cytotoxicity was seldom observed when rabbit, guinea pig, or chicken complements were used in the presence of hyperimmune chicken serum against MSB-1. With duck complement, however, cytotoxicity was always observed. Therefore, duck complement appears to be suitable for assay of hyperimmune chicken serum against MSB-1 cells by the CDAC test.


An infectious bursal disease virus outbreak in 14- and 15-week-old chickens. Infectious bursal disease virus (IBDV) observed in a flock of 14- and 15-week-old chickens was typical of the acute symptomatic IBDV infections more common in younger birds. High flock morbidity was indicated by a marked decrease in feed consumption, although deaths were not excessive. At necropsy, affected birds had small hemorrhages in thigh muscles, creamy-yellow-colored bursae of Fabricius with prominent longitudinal striations, and swollen mottled kidneys. Histopathologic examination revealed bursal lesions typical of IBDV infection. One of six sera from necropsied birds was positive for antibody to IBDV in the agar-gel precipitin (AGP) test, and one week later all 35 samples tested were positive. Bursae were homogenized and found to contain IBDV as evidenced by precipitation, with antibody to IBDV, in the AGP test.


Infectious bursal disease viral infections. I. Complement and virus-neutralizing antibody response following infection of susceptible chickens. Complement (C) titers were decreased at 3 days postinfection, and virus-neutralizing (VN) antibody was detectable at 3 or 4 days postinfection in chickens with infectious bursal disease virus (IBDV). Clotting times were prolonged in all groups tested during the acute phase of the disease. Mortality appeared to be associated with the severity of decrease in C titer. The results suggest that the mortality and many of the clinical signs seen with infectious bursal disease are associated with: 1) a depletion in circulating levels of hemolytic C from the formation of immune complexes at sites of viral replication; and/or 2) a depletion in some clotting factor which results in hemorrhage.


Immunogenetic study on the polymorphism of serum alpha 2-lipoprotein in mink. III. Ld1 allotype of low-density lipoprotein. Mink Ldl antigen of serum low-density lipoprotein was demonstrated by alloantibodies. No genetic relation was found between Ldl and the Lpm system of very-high-density lipoprotein. The existence of an autosomal dominant gene, coding for the new alloantigenic marker, is postulated on the basis of mink breeding data.


Are polyphosphoinositides associated with glycophorin in human erythrocyte membranes? Glycophorin prepared by a lithium di-iodosalicylate-extraction/phenol-partition method was rich in polyphosphoinositides (phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate), but glycophorin extracted by Triton X-100 showed no such enrichment. The enrichment observed in the former preparations appeared not to be caused by pre-existing association between glycophorin and polyphosphoinositides in the human erythrocyte membrane, but to be largely a consequence of the preparative procedures.


Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes. The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].


The involvement of glucocorticoids in regulating the activity of phosphatidate phosphohydrolase and the synthesis of triacylglycerols in the liver. Effects of feeding rats with glucose, sorbitol, fructose, glycerol and ethanol. Feeding rats with sorbitol, fructose, glycerol and ethanol increases the concentration of serum corticosterone without significantly altering the concentration of insulin. This increase appears to be partly responsible for the increases in the hepatic activity of phosphatidate phosphohydrolase (compared with rats fed glucose or 0.9% NaCl) that has been reported [Sturton, Pritchard, Han & Brindley (1978) Biochem. J. 174, 667--670] and the enhanced capacity of the liver to synthesize triacylglycerols. The ethanol-induced increase in phosphohydrolase activity was largely, but not completely, prevented by adrenalectomy.


Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3':5'-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells. The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.


Protein kinase activities in rat pancreatic islets of Langerhans. 1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.


Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. 1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.


Total, latent and active collagenase during the course of post-partum involution of the rat uterus. Effect of oestradiol. 1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.


Effects of diffusible products of peroxidation of rat liver microsomal lipids. The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.


Effects of ethynyloestradiol on the metabolism of [1-14C]oleate by perfused livers and hepatocytes from female rats. Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.


Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine e 3':5'-cyclic monophosphate. Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.


Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin). The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.


Control of hepatic triacylglycerol synthesis. Diurnal variations in hepatic phosphatidate phosphohydrolase activity and in the concentrations of circulating insulin and corticosterone in rats. Male rats were kept for 14 days with alternating 12h periods of light and darkness. The hepatic activity of soluble phosphatidate phosphohydrolase and the concentration of serum insulin were maximum at about 2h after dark. The peak concentration of serum corticosterone occurred 2h before the dark period. It is proposed that corticosterone is partly responsible for the increased phosphohydrolase activity, and that this enables the liver to increase its capacity to synthesize triacylglycerols during the period of maximum feeding.


Poly(adenosine diphosphate ribose) synthetase activity in nuclei of dividing and of non-dividing but differentiating intestinal epithelial cells. Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturing cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono(ADP-ribosylated) protein; 60% of thetotal labelled product was attached to acid-soluble proteins (including histones), and 40% to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADP-ribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.


Evidence for the importance of arginine residues in pig kidney alkaline phosphatase. The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.


Inhibition of rat liver mevalonate pyrophosphate decarboxylase and mevalonate phosphate kinase by phenyl and phenolic compounds. 1. Mevalonate pyrophosphate decarboxylase of rat liver is inhibited by various phenyl and phenolic acids. 2. Some of the phenyl and phenolic acids also inhibited mevalonate phosphate kinase. 3. Compounds with the phenyl-vinyl structure were more effective. 4. Kinetic studies showed that some of the phenolic acids compete with the substrates, mevalonate 5-phosphate and mevalonate 5-pyrophosphate, whereas others inhibit umcompetitively. 5. Dihydroxyphenyl and trihydroxyphenyl compounds and p-chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. 6. Of the three mevalonate-metabolizing enzymes, mevalonate pyrophosphate decarboxylase has the lowest specific activity and is probably the rate-determining step in this part of the pathway.


Steady-state investigations of the mechanism of histidinol dehydrogenase. Initial velocities of the histidinol dehydrogenase reaction (EC 1.1.1.23) were measured as a function of the concentrations of the substrates histidinol and NAD+ and in the presence and absence of the product NADH. The data are consistent with a Bi Uni Uni Bi Ping Pong mechanism. The kinetic constants of this mechanism were determined; Km for histidinol was found to be 14 microM and for NAD+ 0.7 mV; Ki for NAD+ was 0.4 mM.


Isolation and properties of cytochrome c peroxidase from Pseudomonas denitrificans. The isolation of cytochrome c peroxidase, cytochrome c4, cytochrome c-551 and azurin from Pseudomonas dentrificans is described. The peroxidase has a molecular weight of 63,000 and an isoelectric point of 5.6. Its absorption spectrum suggests that it contains two haem c groups/molecule. Preliminary steady-state kinetic data are reported with cytochromes c-551 and c4 and azurin as the second substrate.


Intermediate dehydrogenase-oxidase form of xanthine oxidoreductase in rat liver. A spectrophotometric method for the determination of three forms of xanthine oxidoreductase, namely dehydrogenase (D), dehydrogenase-oxidase (D/O) and oxidase (O), is described. Enzymic fractions obtained from rat liver were found to contain either all three forms, or (under special conditions of preparation) only two forms, D and D/O. The conversion of form D leads to form D/O leads to form O in the presence of Cu2+ ions was shown. Form D/O acted with NAD+ as well as with O2 as electron acceptors, it exhibited greater affinity to NAD+ than to O2, and NAD+ abolished the oxidase activity of this form. Moreover, oxidase activity of form D/O was inhibited by NADH. These facts indicate that NAD+ and O2 compete for the same active site on the enzyme molecule.


Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase. A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.


Collagenase production by synovial fibroblasts treated with phorbol myristate acetate. A series of intracellular events occurring after treatment of rabbit synovial fibroblasts with 0.01 micrograms/ml phorbol myristate acetate (PMA) were measured. Ten minutes after addition of PMA, there was a temporary increase in intracellular cyclic AMP levels, followed by a transient decrease in incorporation of 3H-thymidine into DNA. Approximately 500 ng/mg cell protein of PGE2 were found in culture medium from the 12- to 24-hour incubation period, but significant collagenase was not detectable until 24 to 36 hours. Treatment with aspirin or indomethacin abolished PGE2 production but did not affect collagenase levels. Production of enzyme was associated with a cessation of cell proliferation, measured by protein content/culture and cell number. No enzyme was detectable in untreated cultures. Synovial fibroblasts treated with phorbol myristate acetate may provide a good model for studies on the mechanism of induction of collagenase production.


CLearance of calcium pyrophosphate dihydrate crystals in vivo. II. Studies using triclinic crystals doubly labeled with 45Ca and 85Sr. The clearance rate of isotopically labeled synthetic triclinic calcium pyrophosphate dihydrate (CPPD) crystals injection into rabbit joints was estimated by serial counting. Kinetic analysis using a four compartment model showed that half of the injected dose was cleared from 4 rabbit knee joints in 19.1 +/- 0.42 (SEM) days. Profound hypomagnesemia, produced in 2 rabbits with a low magnesium diet, did not affect the rate of crystal clearance detectably. Lavage of joints with solutions known to promote CPPD crystal solubility failed to remove detectable radioactivity. The previous finding of CPPD crystals in synovial phagocytes by electron microscopy, together with the finding of nuclide activity in the synovium and the failure to remove such activity by joint lavage, suggests that endocytosis by synovial cells is an important, effective mechanism controlling the synovial fluid concentration of crystals in patients with CPPD crystal deposition disease.


[Effects of ampicillin and cefazolin combined with mecillinam (author's transl)]. Combination effects of mecillinam + ampicillin and mecillinam + cefazolin were investigated by the cross diffusion test and by the kinetics of bactericidal action. Synergism was found by cross diffusion in about 50% of strains of E. coli, Klebsiella and Proteus. Sensitivity against ampicillin was correlated with synergism. In mecillinam-resistant strains, synergism is usually absent. The combination of ampicillin or cefazolin with mecillinam leads to bactericidal acceleration; the effects are comparable to those of the combination with gentamycin. Mecillinam seems to be a clinically promising agent for combination chemotherapy. A simple test for synergism is desirable.


Virucidal activity of some quaternary ammonium compounds. Four different quaternary ammonium compounds were tested by microtechniques for evaluating their activity against the type species of six viral groups very frequently involved in human and animal pathology. Three disinfectants showed good activity against five of the representative viruses under test, while the fourth proved less effective. Poliovirus type 1 was found to be completely resistant.


Effect of the diuretic, 1,4-dimorpholino-7-phenylpyrido[3,4-d]-pyridazine (DS-511) and its derivatives on ADH-cyclic AMP system in rat renal medullary slices. The effect of the diuretics, 1,4-dimorpholino-7-phenylpyrido[3,4-d]pyridazine (DS-511) and its 4'-hydroxy derivative [DS-511(4'-OH)] on the ADH-cyclic AMP system was studied in slices of rat renal medulla. These compounds alone did not affect the basal level of cyclic AMP in the slices. Preincubation with 10(-6) mol DS-511 or 10(-7) to 10(-5) mol DS-511-(4'-OH) in the presence of theophylline, inhibited arginine vasopressin stimulated formation of cyclic AMP, but after washing the slices the formation was restored. Etacrynic acid required higher concentrations such as 10(-5) and 10(4-) mol to cause inhibition. No effect was observed with furosemide and hydrochlorothiazide in concentrations up to 10(-4) mol. DS-511(4'-OH) at concentrations higher than 10(-6) mol inhibited the activity of cyclic AMP-phosphodiesterase in the medullary homogenate. These results suggest the water diuretic action of DS-511 is partly mediated by its inhibition of the ADH-cyclic AMP system.


Phenylephrine interaction with specific red cell receptors. An investigation of the action of phenylephrine on the transport of sodium across the human cell membrane is reported. The characteristic parameters of drug-receptor interaction -- the maximal response and ED50 -- were calculated based on the conventional dose-response plot and its linear transformations. The results obtained were in good agreement with other experimental findings.


[Effect of cyproheptadine on the regulation of the adrenal system (author's transl)]. The effect of the antiserotoninergic agent cyproheptadine (C) on the adrenal system was studied in male rats, the adrenocorticol function of which was assessed by analysis of the urinary excretion of free corticosterone. After a feeding period with 0.5--0.7 mg C/kg body weight/day for several months significant differences in the urinary excretion of free corticosterone were only found when the rats were exposed to systemic stress stimuli as cold (increase from 118.7 +/- 5.3 to 305.5 +/- 5.1 vs. 417.8 +/- 48.6 ng/d in the controls; p less than 0.001) and hunger (no increase of free corticosterone in treated animals as compared to an increase to 446.0 +/- 53.6 ng/d in the controls). In contrast to these findings no effect of C was seen when studying the neurotropic stress stimuli reserpine and ether. The same was true when measuring free corticosterone under basal conditions and after ACTH- or metyrapone stimulation. It is concluded that serotonin plays a major role as hypothalamic neurotransmitter in systemic but not in the studied neurotropic stress stimuli. The site of serotonin action does not comprise adrenal synthesis of steroids and the hypothalamic-pituitary-adrenal feedback system, as is shown by the ACTH- and metyrapone stimulation tests.


On the biochemical mechanism of action of 1-propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (HWA 153), a new bronchospasmolytically active methyl xanthine derivative. 1-Propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (HWA 153) is a new bronchospasmolytic agent with a significant influence on the cAMP system of lungs and bronchi. In in vitro experiments HWA 153 inhibits cAMP phosphodiesterase (PDE) isolated from bovine bronchi more than does theophylline. HWA 153 is (in conc. 5 x 10(-4) mol/l) 1.8 and 4.3 times more active as a PDE inhibitor of guinea pig lungs and bronchi, respectively, than theophylline-ethylenediamine. HWA 153 also stabilizes rat erythrocyte membrane against hypoosmotic shock. In isolated guinea pig bronchi HWA 153 (in conc. 5 x 10(-4) mol/l) decreases by 77% bronchial spasm induced by the addition of histamine (5 x 10(-5) mol/l). A significant increase in cAMP level of bronchi was simultaneously observed. In in vivo experiments HWA 153 (25 mg/kg p.o.) inhibits PDE of lungs and bronchi of guinea pigs. Simultaneously, a significant increase in cAMP level in these organs was observed. In in vivo experiments with hypoxic rats, HWA 153 (25 mg/kg p.o.) increases ATP, ATP/ADP ratio and adenylate energy charge (AEC) in hypoxic rats, 1 h after administration. This indicates a positive influence of HWA 153 on the energy metabolism of red blood cells.


The effect of cholestyramine on lipoprotein lipids in patients with primary type IIA hyperlipoproteinemia. The effect of 3 months' treatment with cholestyramine on lipoprotein lipids was investigated in 12 patients. VLDL, LDL and HDL were separated by preparative ultracentrifugation. There was a significant decrease of serum cholesterol and phospholipids and an increase of serum triglycerides. All the VLDL-lipids increased by nearly 30%. The LDL-lipids decreased with a tendency for normalisation of their atypical lipid composition. The small but significant alterations of HDL triglycerides and cholesterol are correlated with the corresponding alterations of the other lipoproteins; the HDL-phospholipids were unchanged. The LDL/HDL-lipid ratios were decreased but not normalised. The 30% decrease of LDL-cholesterol is negatively correlated with an increase in all the VLDL-lipids.


Effects of dipyridamole in experimental atherosclerosis. Action on PGI2, platelet aggregation and atherosclerotic plaque formation. Experimental atherosclerosis in rabbits induced by feeding a standard atherogenic diet for 4 months resulted in an increased sensitivity of platelets to the proaggregatory action of collagen and ADP. Treatment with dipyridamole (3 x 10 mg/day i.m.) for 4 weeks normalized platelet loss in atherosclerotic rabbits and abolished the increased sensitivity to proaggregatory collagen, but not to ADP. Dipyridamole treatment lowered basal as well as PGI2-induced cAMP levels below values seen in platelets from normal rabbits, but the stimulation by PGI2 relative to basal cAMP levels was not affected or even increased by dipyridamole treatment. Dipyridamole did not affect the increased sensitivity of platelets from atherosclerotic rabbits to the antiaggregatory action of PGI2, indicating that dipyridamole decreased absolute cAMP levels, probably due to reduction of the adenine nucleotide pool in platelets without affecting the adenylate cyclase function. Dipyridamole enhanced atherosclerotic plaque formation in arterial walls. Basal as well as PGI2-stimulated cAMP content was lower in homogenates from atherosclerotic than from normal aortic tissue. Dipyridamole-treated animals showed a further decrease in basal as well as PGI2-stimulated cAMP content of the aortic tissue, suggesting that this decrease in cAMP content may be linked to the enhanced proliferative activity seen in artherosclerotic plaque formation.


Characterization of free and tightly bound lipoprotein in intima by thin layer isoelectric focusing. Thin layer isoelectric focusing followed by second dimension quantitative immunoelectrophoresis has been used to characterize the free and tightly bound lipoprotein (lp) fractions in human aortic intima. In 3.3% acrylamide gels plasma low density lipoprotein (LDL) focuses rapidly, but very low density lipoprotein (VLDL) fails to enter the gell and remains at the origin (point of application). Samples of normal intima and lesions were placed directly on the gel and focused for 10--12 h; in normal intima and gelatinous lesions which had not accumulated cholesterol 60--70% of the free LP focused with plasma LDL, but with increasing accumulation of cholesterol the proportion focusing with LDL decreased and the component remaining at the origin increased. In 9 of the 39 samples examined one or more components with pI acid to LDL were present, and in one sample the whole peak focused at a more acid pI. In 4 of 5 lipid-rich centres of gelatinous plaques all the free LP remained at the origin, and the bound LP released by incubation with plasmin remained at the origin in all samples in 3.3% gels although it would focus in 2.6% gels. With antiserum to apo-C  this large LP did not behave like VLDL, and it appears to be an aggregated LDL. An increased proportion of aggregated LDL was associated with (a) accumulation of cholesterol, (b) topographical location towards the centre of plaques, and (c) partial destruction of endogenous LP during incubation of fresh samples of tissue.


Regulation of cholesterol synthesis in skin fibroblasts derived from old people. Sterol synthesis from radioactive acetate and the suppression of this synthesis by human low density lipoprotein (LDL) have been investigated in skin fibroblast strains derived from infant donors and from donors over the age of 70 years. The activity of the enzyme hydroxymethylglutaryl-CoA reductase and its repression by LDL has also been investigated in these fibroblast strains and in senescent cells of the foetal lung cell strain MRC-5. No age-related differences could be detected either in repression of [3H]acetate incorporation by LDL, or in repression of HMG-CoA reductase activity.


High density lipoprotein levels in children of young men with ischaemic heart disease. This study was designed to assess HDL levels in children of young men with IHD, compared with children of asymptomatic men. Like their fathers, sons of patients with heart disease, had significantly lower HDL cholesterols than controls. This difference was independent of fasting triglycerides, obesity, diet or physical activity, and was the only "coronary risk factor" in this young age group.


Virus-specific and anticellular antibodies in molluscum contagiosum. Sera from patients with molluscum contagiosum showed a higher incidence of anticellular and fibrillar anticellular antibodies of IgM class as compared to sera from control subjects. Most patients with anticellular IgM antibodies also had molluscum contagiosum virus-specific antibodies. In a comparative study, there was a higher incidence of anticellular IgM antibodies to normal human epidermis and fibrillar anticellular IgM antibodies in molluscum contagiosum than in warts of psoriasis. The incidence in psoriasis was lower than warts and was similar to that found in control subjects.


[Association of Wilms' tumor, pseudohermaphroditism and glomerular nephropathy. Partial and complete forms of the syndrome]. Four patients, followed at the Buenos Aires Cildren's Hospital are reported: one with a nephrotic syndrome and male pseudohermaphroditism, the second with nephrotic syndrome, male pseudohermaphroditism and Wilms' tumor, the third with pseudohermaphroditism and Wilms' tumor and the fourth with a nephrotic syndrome and Wilms' tumor. The cases presenting nephrotic syndrome, all had an early onset and a rapid and fatal course leading to death in renal failure or secondary to a related bacterial infection. The renal histology in all of the cases with nephrotic syndrome, was very similar: diffuse involvement of all glomeruli consisting in a severe increase of the mesangial matrix, with scarce mesengial proliferation.


Comparative sensitivity of a cultural test and the complement fixation test in the diagnosis of adenovirus ocular infection. The sensitivity of human embryonic kidney (HEK) cell culture and the complement fixation test (cft) in the diagnosis of adenovirus ocular infection has been compared. The optimum time for collection of specimens to obtain the best results by each test has been examined. Sixty-one (53%) of 116 patients with moderate to severe follicular conjunctivitis, clinically indicative of adenovirus infection, had serological evidence of infection or had the virus isolated from ocular swabs. Virus was isolated from 53 patients (45%) and significant antibody rises were found in 33 (27%). When each test was carried out under optimum conditions, virus was isolated from 82% of cases and serological evidence of infection found in 72%. Serological responses as detected by the CFT appeared to be inferior in infections caused by adenovirus type 7 to those encountered in infections due to adenovirus types 3 and 8.


Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy. Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.


An outbreak of adenovirus keratoconjunctivitis in bristol. Nineteen cases of keratoconjunctivitis caused by an adenovirus serologically related to types 10 and 19 are described. Seventeen of the patients presented over a period of 7 weeks and included 4 who were involved in a minor outbreak at a factory. The presentation and clinical features closely resembled those caused by adenoviruses types 8 and 19. Mild to severe follicular conjunctivitis, superficial punctate keratitis, discrete subepithelial opacities, membrane formation, and conjunctival scarring were all observed.


Oxidation of (horse) hemoglobin by copper: an intermediate detected by electron spin resonance. The oxidation of horse hemoglobin by Cu(II) has been followed by the changes in the electron spin resonance spectra of copper. By stopped-flow and freeze-quenching techniques, it is shown that the second-order rate constant for the binding of Cu(II) to hemoglobin is greater than 5 X 10(5) mol-1 s-1 and the apparent first-order rate for the reduction of Cu(II) to Cu(I) is 0.051 s-1. It is also shown that the binding of Cu(II) to hemoglobin is followed by an alteration of the Cu(II) spectrum, decreasing the g values. This process has an apparent rate constant of 17 s-1 and presumably involves a conformational change in the region of the copper binding site. It is also shown that this conformational change is apparently necessary for Cu(II) to oxidize hemoglobin.


Implications of the integrated rate law for the reactions of Paracoccus denitrificans nitrite reductase. The integrated rate law for the reaction of the nitrite reductase of Paracoccus denitrificans, a cytochrome cd, has been established for turnover assays using donor ferrocytochromes c and either nitrite or molecular oxygen as the ultimate acceptor. The time course for the concentration of ferrocytochrome follows the law: formula: (see text), where S is the concentration of donor ferrocytochrome c, So is the initial concentration, t is time, and u1, u2, and u3 are empirical parameters that are constant for a given experiment but depend upon the initial substrate concentration. In particular, all the u1 increase with decreasing initial ferrocytochrome concentration. Saturation of reaction rates at high donor ferrocytochrome concentrations was not observed. The parameter u1 was proportional to the enzyme concentration while u2 and u3 were not. The form of the integrated rate law and the behavior of the u1 impose severe restrictions on possible kinetic schemes for the activity of the enzyme. Contemporary mechanisms that have been proposed for mitochondrial oxidase aa3 are examined and found to be inadequate to explain the reactivity of cytochrome cd. The simplest interpretations of the cytochrome cd data suggest that the enzyme does not bind the ferri and ferro forms of donor cytochromes c with equal affinity and that the enzyme is subject to inhibition by a product of reaction. Eucaryotic horse cytochrome c reacts with the Paracoccus cytochrome cd with 77% of the activity when Paracoccus cytochrome c550 is used as the electron donor.


Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase. We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.


Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions. The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.


Steroidogenic activity of high molecular weight forms of corticotropin. The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules


Copper(II) protoporphyrin IX as a reporter group for the heme environment in myoglobin. Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.


Resonance Raman spectra of cytochrome c oxidase. Excitation in the 600-nm region. The resonance Raman (RR) spectra of oxidized, reduced, and oxidized cyanide-bound cytochrome c oxidase with excitation at several wavelengths in the 600-nm region are presented. No evidence is found for laser-induced photoreduction of the oxidized protein with irradiation at lambda approximately 600 nm at 195 K, in contrast to the predominance of this process upon irradiation in the Soret region at this temperature. The Raman spectra of all three protein species are very similar, and there are no Raman bands which are readily assignable to either cytochrome a or cytochrome a3 exclusively. The Raman spectra of the three protein species do, however, exhibit a number of bands not observed in the RR spectra of other hemoproteins upon exicitation in their visible absorption bands. In particular, strong Raman bands are observed in the low-frequency region of the RR spectra (less than 500 cm-1). The frequencies of these bands are similar to those of the copper-ligand vibrations observed in the RR spectra of type 1 copper proteins upon excitation in the 600-nm absorption band characteristic of these proteins. In cytochrome c oxidase, these bands do not disappear upon reduction of the protein and, therefore, cannot be attributed to copper-ligand vibrations. Thus, all the observed RR bands are associated with the two heme A moieties in the enzyme.


Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to  SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.


Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation. ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.


The orientation of the primary donor in bacterial photosynthesis. The triplet state EPR spectra of magnetically aligned whole cells of Rhodopseudomonas viridis and Rhodopseudomonas palustris display a marked dependence on the orientation of the static EPR field with respect to the alignment field direction. This observation implies that the primary donor species on which the triplets are localized are ordered within the membranes. We have developed a theoretical model for the system to enable calculation of the orientation of the magnetic axes of the primary donor species with respect to the membranes in which they reside. The triplet state spectra are generated by an ensemble of partially ordered magnetic systems and a computer simulation of the experimental results. The triplet orientation is very similar for the two organisms studied, where one axis lies predominantly in the plane of the membrane and the other two axes have approximately equal projections onto the normal to the membrane.


Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26. Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484-501) where computer simulations of the observed triplet state spectra were employed. The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll 'special pair' lies almost entirely along one of the principal magnetic axes of the triplet state. Aso, the 870 nm transition moment makes an angle of approx. 60 degrees with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514-530).


Exchange integral for a variety of tetranuclear ferredoxins. The temperature dependence of EPR spectra of oxidized [4Fe-4S](-1,-2) ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S](-2,-3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm-1; Center S-3 from mung bean, 53 cm-1; the [4Fe-4S](-1,-2) ferredoxin from Thermus thermophilus, 78 cm-1; Center N-2 of NADH ubiquinone reductase, 83 cm-1. Increasing axial distortion in the EPR spectra of the [4Fe-4S](-1,-2), ferrodoxins was associated with higher energy differences. Center N-2, a [Fe-4S](-2,-3) iron-sulfur cluster does not fit this relationship.


Characterization of iron-sulphur centres of plant mitochondria by microwave power saturation. The electron spin relaxation of iron-sulphur centres and ubisemiquinones of plant mitochondria was studied by microwave power saturation of the respective EPR signals. In the microwave power saturation technique, the experimental saturation data were fitted by a least-squares procedure to a saturation function which is characterized by the power for half-saturation (P1/2) and the inhomogeneity parameter (b). Since the theoretical saturation curves were based on a one-electron spin system, it became possible to differentiate between EPR signals of iron-sulphur centres which have similar g values but different P1/2 values. If the difference in the P1/2 values of the overlapped components was small, no significant deviation from these theoretical saturation curves was observed, as shown for the overlapped signals of centre S-3 and the Ruzicka centre of mung bean mitochondria. By contrast, the microwave power saturation data for the g = 1.93 signal (17--26 K) of Arum maculatum submitochondrial particles reduced by succinate could not be fitted using one-electron saturation curves. Reduction by NADH resulted in a stronger deviation. Since the iron-sulphur centres of Complex I were present only in an unusually low concentration in A. maculatum mitochondria, it was proposed that an iron-sulphur centre of the external NADH dehydrogenase contributes to the spectrum of centre S-1. For mung bean mitochondria, the g = 1.93 signal below 20 K could be attributed mainly to centre N-2. The microwave power saturation technique was also suitable for detecting magnetic interactions between paramagnetic centres. From the saturation data of the complex spectrum attributable to centre S-3 and an interacting ubisemiquinone pair in mung bean mitochondria (oxidized state) followed that centre S-3 has a faster electron spin relaxation than the ubisemiquinone molecules. It is noteworthy that the differences in the relaxation rates were maintained despite the interaction between centre S-3 and the ubisemiquinones. Furthermore, a relaxation enhancement was observed for centre S-1 of A. maculatum submitochondrial particles upon reduction of centre S-2 by dithionite. This indicated a magnetic interaction between centres S-1 and S-2.


Intramolecular electron transfer and binding constants in iron hexacyanide-cytochrome c complexes as studied by pulse radiolysis. Internal oxidation and reduction rates of horse cytochrome c in the complexes CII . Fe(III)(CN)6(3)- and CIII . Fe(II)(CN)6(4)-, are 4.6 . 10(4)s-1 and 3.3 . 10(2)s-1, respectively. The binding site of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87 . 10(3) to 2 . 10(3)M-1. The present pulse radiolytic kinetic data is compared with that from NMR, T-jump and equilibrium dialysis studies.


The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles. 1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.


Purification and properties of cytochrome c-553, an electron acceptor for formate dehydrogenase of Desulfovibrio vulgaris, Miyazaki. Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the alpha-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is -0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan. Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c3, but is oxidized by ferricyanide or by O2. Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H2. The formate dehydrogenase does not reduce cytochrome c3 in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.22.-.


Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes. 1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.


A study of the incorporation of cytochrome oxidase into planar synthetic membranes. Cytochrome oxidase molecules were incorporated into black lipid membranes and into a new form of planar synthetic membrane. Studies of these membranes indicated that the incorporation of large membrane bound enzymes into black lipid membranes involves difficulties fundamental to this technique. On the other hand the new method described in this paper is more promising.


Golgi fractions from livers of control and ethanol-intoxicated rats. Enzymic and morphologic properties following rapid isolation. Following rapid isolation, it has been found that Golgi apparatus from ethanol-intoxicated animals contain high levels of galactosyltransferase but also detectable glucose-6-phosphatase and microsomal esterase, as well as 5'-nucleotidase activity. In experiments carried out in parallel on littermate animals but without intoxication, similar recoveries and specific activities of the four enzymes were observed. Morphologic analysis of Golgi fractions isolated from control animals demonstrated no striking morphologic difference to those from the ethanol-intoxicated animals. Indeed, using galloyl glucose-lead staining techniques to mark the lipoprotein particles in situ, it was found that all Golgi apparatus of hepatocytes from control animals were marked by very low density lipoprotein particles. It is therefore concluded that within the limits of the present analyses, Golgi fractions isolated from control animals are as valid as those isolated from ethanol-intoxicated rats.


Interaction of plasma apolipoproteins with lipid monolayers. The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...


Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells. The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI+D+, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI+D-, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI+D- compared to the MGI+D+ clones. The MGI+D- clones produced an unusual polyunsaturated C20:5 fatty acid at 28 degrees C, whereas the MGI+D+ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI+D- clones than of the MGI+D+ clones. The cells of MGI+D+ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.


Pressure effects on lipid-protein interactions in (NA+ + K+)-ATPase. The (Na+ + K+)-stimulated ATPase activity decreases with increasing pressure and a plot of the logarithm of the activity versus pressure shows a change in slope at a defined breakpoint pressure (Pb). The value of Pb increases linearly with increasing temperature. A dT/dP value of 27.7 +- 0.4 (S.D.) K/1000 atm is obtained. This is in very good agreement with the pressure shift for the melting transitions in phospholipids and aliphatic chains. This strongly indicates that an aliphatic chain melting process is involved in the breakpoint in the Arrhenius plot and pressure dependence of (Na+ + K+)-ATPase. The p-nitrophenyl phosphatase activity of this enzyme also decreases with pressure. In this case the plot of the logarithm of the activity versus pressure is linear without a break-point. The temperature dependence for (Na+ + K+)-ATPase was also studied in the presence of fluidizing drugs: desipramine and benzylalcohol. The presence of these drugs had no effect on the inflection point in the Arrhenius plot.


Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells. A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na+ + K+)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4'-diisothiocyano-2,2'-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Free-flow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4'-diisothiocyano-2,2'-disulfonic stilbene-binding proteins. The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.


Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5'-'cap' in viral messenger RNA. Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tesetd in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m7G5' pppA that corresponds to the most simple 'cap' structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G5' pppA or G5' ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm7G). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pm7G carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pm7G was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the 'cap' component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addition of m7GpppA or pm7G when either TMV RNA or phage MS2 RNA was used as an mRNA.


Phosphorlyation of H1 and H5 histones by cyclic AMP-dependent protein kinase reduces DNA binding. Phosphorylation of H1 histones by cyclic AMP-dependent protein kinase may be an important transcriptional control mechanism. We have used affinity chromatography to examine the effect of phosphorylation by this enzyme on the DNA-binding properties of calf thymus H1 histones and two highly basic H1 homologues from condensed and transcriptionally silent nuclei: duck erythrocyte H5 and Strongylocentrotus purpuratus sperm H1. Without in vitro phosphorylation, all three histones were eluted from native DNA-Sephadex G-25 columns at salt concentrations which closely resembled those required to extract these histones from nuclei or chromatin. When a small portion of radioactively phosphorylated histone was chromatographed with untreated carrier histone, the phosphorylated species was consistently eluted from the DNA column at slightly lower salt concentrations than the main histone peak. Rechromatography experiments showed that in vitro phosphorylation of H1 can shift its elution position to lower salt concentrations.


Isoenzymic forms of NAD-linked glycerol-3-phosphate dehydrogenase from rabbit brain. Two major enzyme forms of cytosolic NAD-linked glycerol-3-phosphate dehydrogenase in rabbit brain have been purified to apparent homogeneity. One major enzyme form designated I6.5 exhibits an iso-electric point at pH 6.5, and is indistinguishable from the major form I6.5 found in other tissues. The other major form, designated I5.9, has an isolectric point at pH 5.9, and by amino acid analysis is shown to be a true isoenzyme distinct from form I6.5. Form I5.9 appears to be closely related to or identical with the major enzyme characteristic of heart. Neither the brain enzyme form I5.9 nor the major heart isoenzyme are inhibited by antiserum to the muscle enzyme. Because of the high apparent Km for NADH, it is postulated that the brain isoenzyme I5.9 serves to maintain glycolysis when NADH levels rise under relatively anaerobic conditions especially during fetal and neonatal development.


Ternary complex formation of pig heart lactate dehydrogenase with spin-labelled coenzyme and inhibitors as studied by electron spin resonance. The formation of ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) was studied by means of two NAD derivatives, spin-labelled at N6 and C-8 of the adenine ring. Dissociation constants calculated for the inhibitors oxamate and oxalate from their corresponding ternary complexes are in excellent agreement with data from literature derived from sedimentation experiments. However, the recently postulated enzyme-NADH-sulfite complex was not observed. The mobility of the spin-label, i.e. the protein conformation near the adenine binding pocket in various ternary complexes depends on the type of inhibition or substrate employed.


Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition. Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.


Cyclic nucleotide binding properties and molecular forms of the cyclic-AMP-dependent protein kinase from bovine eye lens. The activity of the cyclic-AMP-dependent protein kinase found in the crude extract of bovine eye lens cortical fibers was increased by 10(-6) M cyclic AMP or 10(-5) M cyclic GMP to the same extent, However, the interaction between cyclic AMP and cyclic GMP in the course of binding to the cyclic-AMP-dependent protein kinase did not seem to be competitive. Scatchard analysis of cyclic GMP binding by the crude extract indicated the presence of two type of cyclic GMP binding sites (Kd1 about 2 . (10(-7) M, Kd2 about 5 . 10(-6) M). Different species of cyclic nucleotide binding fractions were separated by Sephadex G-200 gel chromatography of the crude extract. The bulk of the low affinity cyclic GMP binding activity was found in the exclusion volume. The cyclic-AMP-dependent protein kinase eluted in two fraction (apparent molecular weight 300 000 and 150 000) and both protein kinase fractions were accompanied by the high affinity cyclic GMP binding activity. However, the ratios of this activity to the cyclic AMP binding activity were different in the two fraction, suggesting that different molecular weight forms of the holoenzyme had different cyclic nucleotide binding properties.


Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction. The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.


Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung. 1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.


Pulmonary phosphatidic acid phosphatase. A comparative study of the aqueously dispersed phosphatidate-dependent and membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung. 1. The properties of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase (EC 3.1.3.4) activities of rat lung have been studied in microsomal and cytosol preparations and compared with the properties of the membrane-bound phosphatidate-dependent activities. 2. The microsomal phosphatidic acid phosphatase displayed a prominent pH optimum at 6.5 with a minor peak which varied between 7.5--8 in different experiments. With the cytosol, the major activity was at the higher pH (7.5--8.0) but a distinct optimum was also observed at pH 6.0--6.5. With the membrane-bound substrate, a single broad optimum was observed between pH 7.4 and 8.0 with the cytosol and 6.5--7.5 with the microsomal fraction. 3. Subcellular fractionation studies revealed that the microsomal fraction possessed the greatest proportion of the total phosphatidic acid phosphatase activity and the highest relative specific activity. However, studies with marker enzymes indicated that the aqueously dispersed phosphatidate-dependent activity could be present in plasma membrane, lysosomes and osmiophilic lamellar bodies as well as in the endoplasmic reticulum. 4. The aqueously dispersed phosphatidic acid-dependent activities present in the microsomal and supernatant fractions were inhibited by Ca2+, Mn2+, F- and by high concentrations of Mg2+. In contrast to the membrane-bound phosphatidate-dependent activities, there was little Mg2+ stimulation and only a very slight inhibitory effect was noted with EDTA. A small EDTA-dependent Mg2+ stimulation could be observed with the microsomal fraction but only at the lower pH optimum (6.5). 5. The presence of a number of phosphate esters tended to stimulate rather than inhibit the microsomal activity, indicating that the hydrolase is relatively specific for lipid substrates. Marked inhibitions were noted with lysophosphatidic acid and phosphatidylglycerol phosphate. Phosphatidylcholine produced a slight inhibition. 6. The results indicate that the bulk of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung microsomes and cytosol is not related to the activities observed with membrane-bound phosphatidate. The Mg2+-dependent hydrolase activities may be synonymous. However, unequivocal conclusions will only be possible when the polypeptide or polypeptides responsible for these activities can be purified.


Synthesis and content of ether-linked glycerophospholipids in the harderian gland of rabbits. Although harderian glands are rich in neutral glycerolipids with ether bonds, less than 20% of the choline glycerophospholipids have ether bonds in the white and pink portions of the adult rabbit harderian gland. Only 6% of these are plasmalogens while 94% are alkylacyl glycerophosphocholines. The ethanolamine glycerophospholipids include 37% with ether bonds in both white and pink portions. In the white portion 96% are plasmalogens but only 19% are plasmalogens in the pink portion. The microsomal ethanolaminephosphotransferase (EC 2.7.8.1) is more active with diacylglycerols than with alkylacylglycerols. The microsomal cholinephosphotransferase (EC 2.7.8.2) is equally active with both diradylglycerols. Particularly with microsomes from the pink portion, the apparent Km values for CDPethanolamine and CDPcholine are ower in the presence of alkylacylglycerols than in the presence of diacylglycerols. The incorporation of radioactivity from CDP[14C]ethanolamine and CDP[14C]choline into ethanolamine and choline plasmalogens was increased several-fold by addition of alkylacylglycerols but was not increased substantially by addition of diacylglycerols.


The effect of serum lipoproteins on cholesterol content and sterol exchange in cultured human endothelial cells. Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum. Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.


Regulation of sterol synthesis in human lymphocytes: evidence for post-transcriptional control by low density lipoprotein. The effect of cordycepin (3'-deoxyadenosine), an inhibitor of messenger RNA synthesis, on the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase mediated by lipid-depleted serum was studied in isolated human lymphocytes. 50 micrograms/ml cordycepin, although inhibiting messenger RNA synthesis by more than 50%, had no inhibitory effect on the two and four-fold induction of hydroxymethylglutaryl-CoA reductase when cells were incubated in a medium containing lipid-depleted serum for 8 and 16 h, respectively. This result suggests that newly synthesises messenger RNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in human lymphocytes.


Cell triacylglycerol accumulation from very low density lipoproteins isolated from normal and hypertriglyceridemic human sera. Human fibroblast cells in culture increased their intracellular triacylglycerol levels when exposed to very low density lipoproteins (VLDL) isolated from human plasma. This response was dependent on the amount of VLDL added. VLDL from normal, type IV or type V sera gave similar results. Lipoprotein lipase enhanced this intracellular triacylglycerol accumulation. It was concluded that human fibroblast cells in culture have at least two mechanisms for triacylglycerol uptake from VLDL: (1) uptake from intact lipoprotein either by surface transfer of lipoprotein lipid or internalization of the entire lipoprotein particle, and (2) re-esterification of lower glyceride and fatty acids released by lipoprotein lipase degradation of VLDL.