Activities and some properties of 5'-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine.

1. The maximal activities of 5'-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues. 2. Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K.,from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5. This evidence includes the effects of pH and temperature on the activities of the enzymes. 3. In many tissues, the activities of 5'-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate. In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo. 4. In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase. It is suggested that 5'-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration. 5. The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles. Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles.


Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis.

1. Comparison of the maximum activities of pyruvate kinase with those of phosphofructokinase in a large number of muscles from invertebrates and vertebrates indicates that, in general, in any individual muscle, the activity of pyruvate kinase is only severalfold higher than that of phosphofructokinase. This is consistent with the suggestion, based on mass-action ratio data, that the pyruvate kinase reaction is non-equilibrium in muscle. However, the range of activities of pyruvate kinase in these muscles is considerably larger than that of phosphofructokinase. This difference almost disappears if the enzyme activities from muscles that are known to possess an anaerobic ;succinate pathway' are excluded. It is suggested that, in these muscles, phosphofructokinase provides glycolytic residues for both pyruvate kinase (i.e. glycolysis) and phosphoenolpyruvate carboxykinase (i.e. the succinate pathway). This is supported by a negative correlation between the activity ratio, pyruvate kinase/phosphofructokinase, and the activities of nucleoside diphosphokinase in these muscles, since high activities of nucleoside diphosphokinase are considered to indicate the presence of the succinate pathway. 2. The effect of fructose bisphosphate on the activities of pyruvate kinase from many different muscles was studied. The stimulatory effect of fructose bisphosphate appears to be lost whenever an efficient system for supply of oxygen to the muscles is developed (e.g. insects, squids, birds and mammals). This suggests that activation of pyruvate kinase is important in the co-ordinated regulation of glycolysis in anaerobic or hypoxic conditions, when the change in glycolytic flux during the transition from rest to activity needs to be large in order to provide sufficient energy for the contractile activity. However, lack of this effect in the anaerobic muscles of the birds and mammals suggests that another metabolic control may exist for avian and mammalian pyruvate kinase in these muscles.


Stimulation of triacylglycerol synthesis in rat adipocytes by plasma very-low-density lipoproteins.

The effect of human plasma lipoproteins on lipogenesis from glucose has been studied in isolated rat adipocytes. The very-low-density lipoproteins increased lipogenesis specifically, whereas low-density lipoproteins and high-density lipoproteins were without effect. Such stimulation could be reproduced with partially delipidated very-low-density lipoproteins. Nod-esterified fatty acids and glycerol were also without effect. Pretreatment of the adipocytes with trypsin did not alter the effect of very-low-density lipoprotein. The presence of Ca2+ was required for the full activation of lipogenesis. The synthesis of acylglycerol fatty acids and of acylglycerol glycerol were equally increased. The effect of very-low-density lipoprotein was not additive to that of insulin. It is suggested that very-low-density lipoprotein may directly stimulate lipogenesis in fat-cells, particularly in states when the lipoproteins are present at high concentration in the circulation.


Calcium-dependent inhibition of protein synthesis in rat parotid gland.

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into  trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore  A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187  required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.


Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Characterization by dual-labelling technique and polyacrylamide-gel electrophoresis.

The effect of corticotropin in vivo on total and specific protein synthesis in the adrenal was studied. Adrenal slices from control and corticotropin-treated animals were incubated with [14C]- and [3H]-leucine respectively, followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of subcellular components. With this sensitive dual-labelling technique the following results were obtained. There was a general trophic effect on most adrenal proteins, but corticotropin produced a marked stimulation of a specific adrenal cytosolic protein. This protein has mol.wt. approx. 30 000 and pI 5.5. Corticotropin increased the incorporation of labelled leucine into proteins within 4 h, but no effect was observed before 2 h and after 16 h there was no further increase. These data suggest that this protein is not involved in the corticosteroidogenic action of corticotropin, but rather in the trophic action of this hormone.


Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components.

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.


Effects of glucagon and N6O2'-dibutyryladenosine 3':5'-cyclic monophosphate on calcium transport in isolated rat liver mitochondria.

1. The administration of glucagon to fed rats by intraperitoneal injection, or the perfusion of livers from fed rats with glucagon by the method of Mortimore [Mortimore (1963) Am.J. Physiol. 204, 699--704] was associated with increases of 15- and 5-fold respectively, in the time for which a given load of exogenous Ca2+ is retained by mitochondria subsequently isolated from the liver. This effect of glucagon was (a) also induced by N6O2'-dibutyryl cyclic AMP, (b) completely blocked by cycloheximide, (c) relatively slow in onset (15--60 min) and (d) associated with a stimulation of about 20% in the rates of ADP-stimulated oxygen utilization and Ca2+ transport measured in the presence of succinate. 2. Perfusion of livers with glucagon resulted in the isolation of mitochandria which showed a 50% increase, no significant change and a 40% increase in the concentrations of endogenous Ca, Mg and Pi respectively, when compared with mitochondria isolated from control perfused livers. 3. The administration of insulin or adrenaline to fed rats induced increases of 10- and 8-fold respectively, in the time for which Ca2+ is retained by isolated liver mitochondria. Perfusion of livers with insulin had no effect on mitochondrial Ca2+ retention time. 4. The perfusion of livers from starved rats with glucagon, or the administration of either glucagon or insulin to starved rats, increased by about 2.5- and 15-fold respectively, the time for which isolated mitochondria retain Ca2+. 5. Mechanisms which may be responsible for the observed alterations in Ca2+-retention time are discussed.


Control of rat skeletal-muscle phosphorylase phosphatase activity by adrenaline.

Administration of adrenaline to an isolated rat hindlimb preparation rapidly decreased muscle phosphorylase phosphatase (EC 3.1.3.17) activity and increased heat-stable and trypsin-labile phosphatase inhibitor activity. This was associated with increased tissue cyclic AMP concentrations, phosphorylase (EC 2.4.1.1) activation and glycogen synthase (EC 2.4.1.11) inactivation.


Adenosine 3':5'-cyclic monophosphate production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and [Sar 1,Ala 8]angiotensin II.

Angiotensin II effects on cyclic AMP production and steroid output were studied in a sensitive preparation of isolated rat adrenal glomerulosa cells. With increasing concentrations of angiotensin II logarithmic dose-response curves for aldosterone and cyclic AMP production were similar. The minimum effective dose (0.2nm) for stimulation of aldosterone production also significantly (P<0.001) increased cyclic AMP output. For both aldosterone and cyclic AMP production, the peptide hormone concentration eliciting maximal response (0.2mum) and the ED(50) (median effective dose) values (1nm) were the same; this is consistent with cyclic AMP acting as an intracellular mediator for angiotensin II-stimulated aldosterone production by glomerulosa cells. The angiotensin II antagonist [Sar(1),Ala(8)]angiotensin II inhibited angiotensin II-stimulated corticosterone and aldosterone production in these cells. An equimolar concentration of antagonist halved the response to 20nm-angiotensin II, and complete inhibition was observed with 0.2mum-antagonist. In contrast, [Sar(1),Ala(8)]angiotensin II had no effect on maximally stimulated steroidogenesis induced by serotonin and a raised extracellular K(+) concentration. Increasing concentrations of [Sar(1),Ala(8)]angiotensin II alone decreased corticosterone and aldosterone outputs significantly (P<0.05) at concentrations of 20nm and 2nm of antagonist respectively. A significant (P<0.001) decrease in cyclic AMP production occurred with 2mum antagonist and this was comparable with the decrease in aldosterone production. It is concluded that [Sar(1),Ala(8)]angiotensin II can independently affect glomerulosa-cell steroidogenesis, possibly by modulating adenylate cyclase activity.


Inhibition by calcium ions of adenosine cyclic monophosphate formation in sealed pigeon erythrocyte  'ghosts'. A study using the photoprotein obelin.

1. Sealed pigeon erythrocyte 'ghosts' were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The 'ghosts' were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed 'ghosts', containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)--10(4) obelin luminescence counts/10(6) 'ghosts', which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these 'ghosts' was 30--50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the 'ghosts' resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the 'ghosts' and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the 'ghosts' was markedly decreased by sealing EGTA inside the 'ghosts'. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte 'ghosts' could be inhibited by more than 50% by free Ca2+ concentrations in the range 1--10 micrometer.


Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1  of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.


Effect of intessive plasmapheresis on the plasma cholesterol concentration with familial hypercholesterolemia.

Plasmapheresis was studied as a means of reducing the serum cholesterol concentration in 3 hypercholesterolemic patients who each underwent courses of intensive plasmapheresis with removal of 250--500 ml of plasma each day for 5--9 days. In one homozygous Type II patient, the serum cholesterol concentration decreased from 609 +/- 45 mg/100 ml (mean +/- SEM) to 365 +/- 17 mg/100 ml (40% decrease, P less than 0.05) with two different courses of plasmapheresis. In the two other patients with non-homozygous hyperbetalipoproteinemia the serum cholesterol concentration decreased from 289 +/- 27 mg/100 ml to 205 +/- 19 mg/100 ml (29% decrease, p less than 0.05). After cessation of treatment, the cholesterol concentration returned to pre-treatment levels in 10--13 days in the homozygous patient and 7 days in one non-homozygous hyperbetalipoproteinemic patient; clofibrate (2 g/day) in this patient was associated with a smaller reduction of the cholesterol concentration with plasmapheresis and an increased rate of return of pre-treatment levels after plasmapheresis was stopped. Sustained plasmapheresis for 6 days in the other non-homozygous hyperbetalipoproteinemic patient resulted in a new approximate "steady state" with a serum cholesterol concentration of 176--199 mg/100 ml compared  with a pre-plasmapheresis value of 227 mg/100 ml. The response of the plasma cholesterol levels to plasmapheresis was subjected to kinetic analysis based on a current model of the regulation of lipoprotein metabolism.


Glycosaminoglycan-lipoprotein complexes from aortas of hypercholesterolemic rabbits. Part 1. Isolation and characterization.

Glycosaminoglycan-lipoprotein complexes were isolated from rabbit aortas exhibiting nearly confluent cholesterol-induced foam cell lesions by extraction with 0.15 M NaCl. Purification and characterization was achieved by gel chromatography, non-ionic differential flotation and by cellulose polyacetate electrophoresis. Analysis showed that these complexes consisted of very low density lipoproteins, heparan sulfate, chondroitin sulfate-C and hyaluronic acid. The demonstration that rabbit intimal foam cell lesions contain  extractable glycosaminoglycan-lipoprotein complexes makes this animal model an excellent tool for further studies on the role of these complexes in the atherogenic process.


Efficacy of hypolipidemic treatment in inhibition of experimental atherosclerosis: the effect of nicotinic acid and related compounds.

The hypolipidemic and antiatherogenic effects of different nicotinic acid derivatives were studied. Five rabbit groups maintained on an atherogenic diet were given simultaneously various nicotinic acid derivatives (50 mg/kg body weight/day): nicotinic acid, Xantinol-nicotinate, beta-pyridylcarbinol or Pirozadil (bis-3,4,5-trimethoxybenzoate, 2,6-pyridindiyldimethylene). All 4 compounds showed a clear hypocholesterolemic and antiatherogenic effect, as measured by serum cholesterol, and by planimetric evaluation of the aortic lesions in terms of percent surface area affected in the aortas and coronary lumen. The simultaneously observed elevation of the HDL/LDL cholesterol ratio of the aortic tissue possibly indicates an antiatherogenic effect of these changes.


Association of structural glycoproteins with lipids or lipoproteins.

Gradient density ultracentrifugation was used to assess the lipid binding of 125I-labelled structural glycoproteins and standard proteins. The results show that solubilized structural glycoproteins from cartilage or aorta will associate spontaneously with lipids or lipoproteins. In addition, structural glycoprotein preparations from tissues which have not been delipidated contain a fraction which is already associated with lipid. Standard proteins do not behave in the same way. It is proposed that structural glycoprotein-lipid binding may be one of the mechanisms involved in the intercellular deposition of plasma lipoproteins in atherosclerotic arteries.


Dose-response study of bezafibrate on serum lipoprotein concentrations in hyperlipoproteinanemia.

The effect of bezafibrate in the dosages 450, 900 and 1350 mg daily on serum lipoprotein concentrations were studied in 20 subjects with primary hyperlipoproteinaemia (16 of type IV, 2 of type II B, 1 of type III). Except for LDL cholesterol maximum effects were obtained with 450 mg daily. Total serum cholesterol and triglycerides (TG) fell significantly. Mean very low density lipoprotein (VLDL) TG fell by 54%. The maximum effect on low density lipoprotein was obtained with 900 mg daily. The effect was highly dependent on initial concentrations, decreases being observed above 4 mmoles/l and increases below that concentration. High density lipoprotein cholesterol increased by 31% (P less than 0.01) independently of the VLDL decrease. Three subjects suffered gastrointestinal side-effects on 1350 mg daily. Only benign reversible changes were noted on non-lipid measurements. Bezafibrate is a well-tolerated drug with a good VLDL TG lowering effect. It is particularly effective in increasing HDL cholesterol concentrations.


One-year study of the effect of bezafibrate on serum lipoprotein concentrations in hyperlipoproteinaemia.

The effect of bezafibrate (2-(4-[2-(4-chlorobenzamido)-ethyl]-phenoxy)-2-methylporopionic acid) (450 mg daily) on serum lipoprotein concentrations was studied for 1 year in 14 subjects with hyperlipoproteinaemia (3 of type II A, 2 of type II B, 2 of type III and 7 of type IV). After 2 months serum cholesterol decreased from 7.12--6.08 mmoles/l (15% P less than 0.01), triglycerides from 2.52--1.96 mmoles/l (22%, P less than 0.01), very low density lipoprotein triglycerides from 1.48--0.86 mmoles/l (42%, P less than 0.01) and low density lipoprotein cholesterol from 5.00--3.81 mmoles/l (24%, P less than 0.01). While triglyceride concentrations of serum and lipoproteins remained constant for 1 year, serum and LDL cholesterol concentrations increased slightly after 6 and 12 months. The former rise was partly due also to a rise of the possibly beneficial high density lipoprotein cholesterol from 1.27--1.68 mmoles/l (32%, P less than 0.01) after 6 months, making the effect on total cholesterol less pronounced. Subjective side-effects were nausea in one patient. No severe biochemical side-effects were noted.


The action of human high density lipoprotein on cholesterol crystals. Part 1. Light-microscopic observations.

High density lipoprotein (HDL) was found in vitro to form myelin buds (liposomes) from washed crystals of free cholesterol (commercial or atheroma sources). This activity led to the progressive destruction and solubilization of the crystals. Low density and very low density lipoproteins did not have any effect. Liposome formation and solubilization were accelerated by calcium ions, phospholipase A and polyunsaturated lecithin (Lipostabil). Cholesterol crystals were nearly completely destroyed after 18 h incubation with HDL-Lipostabil.


The action of human high density lipoprotein on cholesterol crystals. Part 2. Biochemical observations.

Electron microscopy of the reaction product between human pooled high density lipoprotein (HDL) and cholesterol shows that characteristic liposome macromicellar bodies are formed. These bodies vary in size between 30 and 1200 nm. In comparison with HDL, they contain markedly more cholesterol, but less protein and phospholipid. Their phospholipid pattern shows enrichment with sphingomyelin and phosphatidyl serine in comparison with HDL.


Potentiation of isoprenaline-induced plasma cyclic AMP response by aminophylline in normal and asthmatic subjects.

1 The effect of intravenous aminophylline infusion (6 mg-1 kg in 20 min, then 0.9 mg-1 kg-1 h thereafter) and its interaction with inhaled isoprenaline (800 microgram ex inhaler) on plasma cyclic AMP levels was studied in five normal and five asthmatic subjects. 2 Aminophylline infusion alone did not significantly change plasma cyclic AMP levels in either group. 3 In both groups, the plasma cyclic AMP response to isoprenaline aerosol inhalation was enhanced by aminophylline, although to a lesser degree in the asthmatic subjects. 4 The results provide in vivo evidence consistent with the hypothesis that in man, therapeutic doses of aminophylline may exert their clinical effects by inhibition of cyclic AMP phosphodiesterase.


Chemical modification and immunogenicity of membrane fractions from mouse tumour cells.

A crude membrane fraction isolated from mouse tumour cells was treated with various chemicals. The effects on the immunogenicity of the membrane sample were tested in syngeneic mice for tumour protection, using a challenge dose of 10(5) viable tumour cells. Best protection was obtained after immunization of mice with a membrane sample modified with dimethylsulphate. Up to 60% of the animals remained tumour free, and the tumour-bearing animals showed a greatly increased mean survival time. The post-challenge sera contained no detectable amounts of cytotoxic antibodies. The membrane sample isolated from tumour cells which had been modified with dimethylsulphate showed less immunogenicity than the modified cells or the membrane fraction from unmodified cells.


ESR study of development of RFM/Un murine myeloid leukaemia.

The blood, spleen and liver of RFM/Un mice were examined by means of electron spin resonance (ESR) throughout the course of myeloid leukaemia induced by i.v. injection of leukaemic spleen cells. Marked changes in the concentration of iron transferrin and caeruloplasmin were observed in the blood 1 day after injection. As the disease progressed, changes occurred in the concentrations of the ascorbyl radical and of paramagnetic metal complexes in both spleen and liver. These changes are compared with those induced in RF/J mice injected with normal and leukaemic spleen cells from RFM/Un mice.


Paramagnetic changes in cancer: growth of Walker 256 carcinoma studied in frozen and lyophilized tissues.

Samples of Walker 256 carcinoma grown in muscles of Sprague-Dawley rats were studied at low temperatures before and after lyophilization. The effects of lyophilization on the ESR spectra were different for tumours and normal muscle. Prior to lyophilization of a tumour sample, there was a decrease in free radicals, while after the lyophilization, there was an "increase". The "increase" was due to the lyophilized tumour having a narrower line, producing a greater peak-peak height measurement than in muscle, without an increase in the total number of spins. Exposure of lyophilized samples to air produced an increase in the intensity of the spectra and a change in line shape; also these effects differed for tumour and muscle. Mn++ levels were lower in tumour than in muscle, a difference eliminated by lyophilization. Poor growth conditions in tumours increased the occurrence of ESR spectra due to NO complexes with both heme and non-heme iron proteins. These results may help to resolve the principal controversies about experimental findings in ESR of tumours. At least part of the signals seen after lyophilization do not reflect free radicals in vivo. The signals after lyophilization reflect biochemical differences between tumour and muscle; spectroscopic data indicate that it is feasible to determine the molecular basis of these differences.


C-type virus particles in human urogenital tumours after heterotransplantation into nude mice.

C-type viruses were formed in heterotransplants of 5/14 human urogenital tumours which had been serially transferred in nude mice of NIH(S) background. Except for one case in which C-type particles were present in the epithelial cells as well as the connective tissue, the viruses were only found within the stroma of the heterotransplanted tumours. Peroxidase labelling with anti-mouse serum demonstrated that the connective tissue supporting the transplanted human tumours was of mouse origin. Competition radioimmunoassays demonstrated that MuLV interspecies viral protein was present in high titre in the transplanted tumour extracts and also in extracts of 2 spontaneous mouse-tumour extracts. These data suggest that endogenous viruses of the nude mice are activated by the graft, and only subsequently infect the human tumour cells and form particles.


ACTH-like activity in immune complexes of patients with oat-cell carcinoma of the lung.

Immune complexes could be isolated from sera of 7 patients with oat-cell carcinoma of the lung, but not from 5 normal controls, using zonal ultracentrifugation. After ultracentrifugation, fractions containing macromolecular IgG were absorbed on a protein A-sepharose column and the immune complexes were eluted and dissociated by glycin-HCl buffer at pH 3.5. The eluates were tested for the presence of tumour-associated proteins as carcinoembryonic antigen (CEA), non-specific crossreacting antigen (NCA), alpha2 pregnancy associated antigen (alpha2PAG) and isoferritin. Whereas none of these tumour-associated antigens could be demonstrated, an ACTH-like activity was detected in the immune-complex fractions of 4 patients with oat-cell carcinoma, by radioimmuno- and bioassay. Polyacrylamide electrophoresis of an immune-complex fraction from a patient with Cushing syndrome showed ACTH-like activities, with mol. wt of 110,000, 75,000, 30,000 and less than 20,000 (all glycoproteins) indicating the presence of different subfractions of big ACTH.


Significance of the proline assay in the study of anti-MSV cell-mediated immune reactions.

The cytolysis of 3H-proline-labelled tumour cells growing in monolayer by syngeneic immune lymphocytes has been studied in the murine sarcoma virus (MSV) system. Results show that the proline assay (PA) is a convenient way to reveal the activity of cytolytic T lymphocytes against FMR-like antigens. Using the same effector and target cells, the classical chromium-release test (CRT) fails to reveal any cytolytic activity, and the visual microcytotoxicity assay as well as several derived isotopic methods are known to reveal mainly non-specific reactions due to non-T effector cells. The PA, therefore, appears to be a useful method for testing an antitumour reaction against tumour cells in monolayer. The results are, however, different from those obtained in the CRT using the same effector cells but lymphoma cells in suspension as targets, the major discrepancies being the following: (a) the PA does not provide truly quantitative data, due to the very high lymphoid: effector cell ratios needed in this test; (b) unexpected patterns of antigenic specificities are sometimes detected in PA; (c) a non-specific natural killer activity of non-T cells is frequently detected in the PA, masking at low lymphoid: target cell ratios the T-dependent specific cytolysis; (d) the H-2 restriction of the cytolytic T-cell activity is poorly detected in PA, whereas the role of H-2 antigens is clearly shown by blocking experiments using anti-H-2 antibodies.


Malate dehydrogenase. Kinetic studies of substrate activation of supernatant enzyme by L-malate.

At pH 8.0 in 0.05 M Tris-acetate buffer at 25 degrees C, homogeneous supernatant malate dehydrogenase exhibits substrate activation by L-malate. The turnover number, Michaelis constant for L-malate, and Michaelis constant for NAD are: 0.46 X 10(4) min(-1), 0.036 mM, and 0.14 mM, respectively, for nonactivated enzyme and 1.1 X 10(4) min(-1), 0.2mM, and 0.047 mM for the same series of constants in activated enzyme. Nonactivating behavior is observed at concentrations between 0.02 and 0.15 mM L-malate and activating behavior is observed between 0.15 and 0.5 mM L-malate. L-Malate activation is compared with similar activation of mitochondrial malate dehydrogenase. While it is not possible to exclude unequivocally all mechanisms, the data seem to be consistent with the occurrence of a fundamentally ordered bi bi mechanism, possibly involving activation through the allosteric binding of L-malate. It is concluded that the data are consistent with a form of the "reciprocating compulsory order mechanism" in which nonactivated enzyme reflects catalysis by one subunit and activated catalysis expresses the coordinated activity of two subunits. The allosteric interaction and the "reciprocating mechanism/ are not mutually exclusive.


Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.


Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril.

The use of the iodinating reagent 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (chloroglycoluril) to selectively label membrane surface proteins was investigated with the following systems: enveloped viruses (Sendai and Newcastle disease viruses), human erythrocytes, and nucleated cells propagated both in suspension (EL-4) and in monolayer culture (BHK-21). Conditions are described for specifically iodinating surface proteins while maintaining full virus integrity or cell viability. Comparison of the chloroglycoluril method with the lactoperoxidase and chloramine-T methods for labeling surface membrane proteins shows that the chloroglycoluril method has a number of advantages: It routinely produces a 3- to 17-fold greater specific radioactivity without sacrificing viral or cellular integrity, it is technically simpler to use, it does not require the addition of extraneous protein to initiate the reaction nor a strong reducing reagent to terminate it. Chloroglycoluril also proved to be an effective substitute for chloramine-T in the nonvectorial labeling of viral and cellular proteins. Membrane protein samples were solubilized with the detergent sodium dodecyl sulfate before iodination or labeled in the presence of high iodide concentrations without prior solubilization. The resulting specific radioactivities generated by the use of chloroglycoluril were equal to or greater than those generated by the chloramine-T method. The effectiveness, simplicity of use, and versatility of chloroglycoluril recommend it as an iodinating reagent for both surface-specific and nonvectorial labeling of membrane systems.


Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.


Subunit dissociation in the allosteric regulation of glycerol kinase from Escherichia coli. 2. Physical evidence.

The dependence of the molecular weight of glycerol kinase on enzyme concentration and on binding of fructose 1,6-bisphosphate has been examined by velocity sedimentation, gel filtration, and polyacrylamide gel electrophoresis. The sedimentation coefficient and Stokes radius decrease as a consequence of dilution in a manner consistent with dissociation into half-molecules, with limiting values suggesting molecular weights of about 218,000 and 136,000 for the associated and dissociated species, respectively. Fructose 1,6-bisphosphate (5 mM) prevents the decrease in sedimentation coefficient brought about by dilution, suggesting a decrease in the apparent subunit dissociation constant of at least four orders of magnitude. Electrophoretic mobility in polyacrylamide gels increases as a consequence of dilution in the absence, but not in the presence, of fructose 1,6-bisphosphate. Ferguson plots indicate that glycerol kinase has the same molecular weight in the presence of fructose 1,6-bisphosphate as the covalently cross-linked tetramer and is substantially smaller in the absence of fructose 1,6-bisphosphate. These results are consistent with the model of glycerol kinase proposed in the preceding paper of this issue [de Riel, J.K., and Paulus, H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding.


Nicotinamide adenine dinucleotide dependent isocitrate dehydrogenase from beef heart: subunit heterogeneity and enzyme dissociation.

Heterogeneity in the subunits of nicotinamide adenine dinucleotide dependent isocitrate dehydrogenase from beef heart mitochondria was investigated using one- and two-dimensional electrophoretic analyses in polyacrylamide gels. Electrophoresis under nondenaturing conditions, at several values of pH and gel concentration, followed by second-dimension electrophoresis in the presence of sodium dodecyl sulfate showed that the active enzyme contains four different subunits. The details of these two-dimensional patterns, reelectrophoresis of the active enzyme band under nondenaturing conditions, together with additional evidence indicate that under certain nondenaturing conditions the enzyme exists partially dissociated into its subunits. The molecular weights of the four subunits, determined from electrophoretic mobilities obtained in the presence of sodium dodecyl sulfate, were different, varying between 39 000 and 41 300. Tryptic peptide maps of the subunits are substantially different.


Generation of a free alpha-amino group by Raney nickel after 2-nitro-5-thiocyanobenzoic acid cleavage at cysteine residues: application to automated sequencing.

The selective reaction of SH containing proteins and peptides with NTCB (2-nitro-5-thiocyanobenzoic acid) has been reported (Degani, Y., & Patchornick, A. (1974) Biochemistry 13, 1; Jacobson, G.A., Schaffer, M.H., Stark, G.R., & Vanaman, T.C. (1973) J. Biol. Chem. 248, 6583). With this reagent, cysteinyl peptide bonds are selectively cyanylated and subsequently cleaved under alkaline conditions. In the present study we have successfully cleaved the beta-chains of guinea pig hemoglobin at the single cysteine and the peptides thus obtained were separated. However, the C-terminal peptide was blocked at its N terminal by a thiazolidine ring and hence could not be used for Edman degradation sequence analysis. Deblocking of this peptide was successfully done by Raney nickel in the buffer medium of pH 7.0, and also in water, at 50 degrees C for 6 to 10 h. The Raney nickel reagent is used in large excess by weight (at least ten times the weight of sulfur compound) over the compound to be desulfurized. Under these conditions, control experiments on cysteine, methionine, and some other amino acids showed that only the sulfur containing amino acids are degraded by Ni(H). Cysteine and methionine were rapidly converted to alanine and beta-aminobutyric acid, respectively. Gel electrophoresis of the iminothiazolidine peptide after exposure to Ni(H) showed no breakage of the chain.


Resonance Raman spectra of whole mitochondria.

The resonance Raman spectra of reduced cytochromes b and c and cytochrome oxidase in whole mitochondria have been recorded without any instrument modifications. The contributions of the individual cytochromes have been identified by comparison with the characteristic features observed in partially purified preparations including: (i) the strong dependence of the intensity patterns on excitation wavelength relative to the peak positions of the alpha, beta, and gamma absorption bands of the cytochromes; and (ii) the presence of marker bands for heme type. Since the Raman spectra can be used as an intrinsic indicator of interaction between hemes, the ability to record spectra in intact mitochondria opens the possibility to study heme-heme interactions in the functioning membrane in situ.


Cell-free synthesis of herpes simplex virus DNA: the influence of polyamines.

The effect of polyamines on cell-free DNA synthesis of herpes simplex virus DNA in two different systems is investigated. Purified nuclei from infected cells are devoid of measurable amounts of putrescine, spermidine, and spermine, while an unfractionated lysate contains the polyamines at close to their respective cellular concentrations. Spermine, 0.3 mM, and 0.5 mM spermidine, when added to the nuclear system, decrease the extent of viral DNA synthesis to the level found in the lysate system, the size of the cell-free viral DNA product is increased, and a specific inhibition of repair-type DNA synthesis is observed. These effects of the polyamines occur only in the presence of ATP and not the other three ribonucleoside triphosphates.


Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.


Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase.

The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.


Dependence of boundary lipid on fatty acid chain length in phosphatidylcholine vesicles containing a hydrophobic protein from myelin proteolipid.

Lipophilin, a hydrophobic protein fraction, purified and delipidated from the proteolipid of human myelin, possesses a layer of boundary lipid surrounding it when incorporated into lipid vesicles. The protein reduces the energy absorbed during the lipid phase transition, indicating that the boundary lipid does not go through the phase transition. The amount of boundary lipid was estimated by plotting the enthalpy of the transition against the protein to lipid mole ratio and extrapolating to deltaH = 0 for a number of synthetic phosphatidylcholines, to determine the ability of fatty acid chains of varying length to interact with the protein. The amount of boundary lipid was found to be similar, 21-25 molecules per molecule of lipophilin, for fatty acid chains of length 14-18 carbons but somewhat less, 16 molecules of lipid per molecule of protein, for a fatty acid chain length of 12 or for one with a trans double bond (18:1tr). No preferential interaction was observed with a lipid containing a particular fatty acid chain length when the protein was incorporated into a mixture of these lipids. These results suggest that the binding of lipids to the boundary layer of other membrane proteins and enzymes may not depend significantly on lipid fatty acid chain length.


The role of arginine residues in the function of D-glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of apo-glyceraldehyde-3-phosphate dehydrogenase from rat skeletal muscle in the presence of butanedione is the result of modification of one arginyl residue per subunit of the tetrameric enzyme molecule. The loss of activity follows pseudo-first-order kinetics. NAD+ increases the apparent first-order rate constant of inactivation. The effect of NAD+ on the enzyme inactivation is cooperative (Hill coefficient = 2.3--3.2). Glyceraldehyde 3-phosphate protected the holoenzyme against inactivation, decreasing the rate constant of the reaction. At saturating concentrations of substrate the protection was complete. The Hill plot demonstrates that the effect is cooperative. This suggests that subunit interactions in the tetrameric holoenzyme molecule may affect the reactivity of the essential arginyl residues. In contrast, glyceraldehyde 3-phosphate had no effect on the rate of inactivation of the apoenzyme in the presence of butanedione. 100 mM inorganic phosphate protected both the apoenzyme and holoenzyme against inactivation. The involvement of the microenvironment of the arginyl residues in the functionally important conformational changes of the enzyme is discussed.


Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.


Phospho-N-acetylmuramoyl-pentapeptide-transferase of Escherichia coli K12. Properties of the membrane-bound and the extracted and partially purified enzyme.

Phospho-N-acetylmuramoyl-pentapeptide-transferase (UDP-N-acetyl-muramoyl-L-alanyl-D-gamma-glutamyl-L-lysyl-D-alanyl-D-alanine:undecaprenoid-alcohol-phosphate-phospho-N-acetylmuramoyl-pentapeptide-transferase, EC 2.7.8.13) was solubilized by repeated freezing and thawing of crude envelopes of Escherichia coli K12. The solubilized enzyme was partially purified by gel filtration and ion-exchange chromatography. This preparation contained small amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol but no endogenous lipid substrate, C55-isoprenyl phosphate, could be detected. Some catalytic properties (exchange reaction) of the solubilized enzyme were compared to those of membrane-bound transferase. The transfer activity of the partially purified transferase was restored by the addition of an aqueous lipid dispersion. All the transferase activity was found to become incorporated into the liposomes. Preincubation of the transferase preparation with phospholipase A2 or D strongly reduce both exchange and transfer activity. This suggests that phospholipids sensitive to phospholipases are necessary for the enzymatic reaction. Different effects of some neutral detergents on the exchange activity were reported.


Activation of mammalian cyclic AMP phosphodiesterases by trypsin.

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.


Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase.

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.


Nitrogenase X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with Mössbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the Mössbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the Mössbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.


Electron nuclear double resonance of cytochrome oxidase: nitrogen and proton ENDOR from the 'copper' EPR signal.

Proton ENDOR resonances have been found from at least two different protons with fairly large and isotropic couplings of about 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H2O. We have obtained nitrogen ENDOR from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have not yet demonstrated experimentally ENDOR characteristic of the copper nucleus itself.


Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.


Isolation and partial characterization of secreted mouse pituitary prolactin.

Prolactin secreted by mouse anterior pituitaries was purified by gel filtration on Sephadex G-100. Electrophoretic homogeneity of the purified hormone was demonstrated in several gel systems, and electrophoresis in the presence of sodium dodecyl sulfate indicated an apparent molecular weight of 21 000 +/- 2000. Mouse and ovine prolactin displayed parallel dose vs. response curves in radio-receptor binding studies, indicating that these two hormones compete for identical receptor sites on rabbit mammary membranes. Comparative peptide mapping studies carried out on tryptic digests of mouse and ovine prolactin suggested only partial homology between the two hormones. Internally labeled monomeric mouse prolactin was observed to undergo aggregation following storage at --20 degrees C for 2 months.


Properties of adenylate kinase after modification of Arg-97 by phenylglyoxal.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) isolated from porcine skeletal and heart muscle and from rabbit muscle are inactivated when a single arginine residue is modified. In adenylate kinase from pig the modified residue was identified as Arg-97 by peptide-mapping. In native adenylate kinase Arg-97 is located at the bottom of the active site cleft. The protein fluorescence of modified adenylate kinase is reduced. Whereas the addition of AMP, ADP and MgATP quench the fluorescence of native adenylate kinase, the fluorescence of phenylglyoxal-modified adenylate kinase is only affected by ADP and MgATP. This finding is discussed in connection with the structural isomerization observed in native adenylate kinase by X-ray diffraction analysis.


The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.


Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.


Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: a possible role in co-carcinogenesis.

In recognition of the co-carcinogenic effects of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzanthracene and dependent upon adsorption to silica and upon the surface density of benzanthracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzanthracene from the silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates. The rate of benzanthracene uptake by dipalmitoyl-L-alpha-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its solubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzathracene molecules are dispersed uniformly on the silica surface. Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed  benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles. These results indicate that particulates can enhance the cellular availability of the chemical carcinogens.


On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart.

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action  of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.


Properties of epinephrine-induced activation of cardiac adenosine 3',5'-monophosphate-dependent protein kinase.

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (-cyclic AMP/"cyclic AMP) of 12 000 X g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.


Glucosyl transferase activity of bovine galactosyl transferase.

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.


Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins.

32P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E1 one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP. Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes. These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.


Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast. Role of cyclic AMP.

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.


ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers.

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester (I) and 1 : 14 ester (II). The nitroxide label of I is aligned in the membrane with a very large angle of tilt (47 degrees +/- 1.5 degrees) with respect to the normal to the membrane surface; II does not show such a tilt. I gives spectra corresponding to immobilized label while II gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in I and II are not present at the phosphatidylcholine-water interphase. The data supports a 'horseshoe' configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.


Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase.

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 . 10(5) or 6.0 . 10(5) molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4-diisothiocyano-1,2-diphenyl-ethane-2,2'disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.


Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.


Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase.

Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor.


Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction.

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.


Purification and characterization of (Na+ + K+)-ATPase from toad kidney.

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.


Leukocyte migration inhibitory factor: a serine esterase released by stimulated human lymphocytes. Kinetic analysis and inhibition by cyclic GMP.

Lymphocytes, stimulated with concanavalin A, release small amounts of non-immunoglobulin, highly reactive proteins called lymphokines. One of these, a serine esterase, termed leukocyte migration inhibitory factor according to its function in vitro, is found in supernatants of stimulated human lymphocytes at concentrations less than 1 ng/ml. The esterase was purified in good yield and its esterolytic activity was measured by a sensitive radioenzymic assay. The kinetics of the esterolytic activity were studied and the effect of various nucleotides examined. Competitive inhibition of esterolysis was seen with cyclic GMP at concentrations down to 10(-7) M, and with 2',3'-cyclic CMP at a concentration of 10(-3) M. A role of this esterase, not only as a mediator acting upon polymorphonuclear leukocytes, but also as an intracellular regulator of lymphocyte activation, is discussed.


Ribonucleic acid synthesis in rat liver during fatty acid stimulated secretion of very low density lipoproteins.

Production of very low density lipoproteins by the liver depends on the cellular availability of fatty acids. It is stimulated by the uptake of free fatty acids from the plasma and by increased lipogenesis and is inhibited by actinomycin D, suggesting that RNA synthesis is involved in the regulation of apolipoprotein synthesis. This hypothesis has been investigated in rats in vivo and in isolated perfused livers with and without stimulation by fatty acid overload: [14C] orotate incorporation in liver polyribosomal RNA is 60 per cent greater in stimulated livers as compared to controls. This increase is primarily due to a higher incorporation in bound polysomes and in those containing at least six ribosomes and does not result from the inhibition of ribonuclease. RNase digestion of polysomal RNA (4.10(-10) M enzyme, 0 degrees C, 3 h) shows that there is twice as much radioactivity in the hydrolyzed RNA of stimulated livers as compared to controls. After partial purification of poly A-rich RNA by affinity chromatography, the mass yield and radioactivity are increased by 100 per cent in stimulated livers as compared to controls. In conclusion, de novo RNA synthesis seems to be necessary for fatty acid stimulation of VLDL production.


[Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens].

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.


Characterization of the cyclic AMP-dependent protein kinase from rat pancreas, further purification of the catalytic subunit, substrate specificity, effect of the pancreatic heat stable inhibitor.

In order to investigate the sequence of events triggered by cyclic AMP and cyclic GMP in exocrine pancreatic cells, the identification of the various protein kinases possibly present in this tissue is of major interest. Further analysis of the two cyclic AMP-dependent protein kinases previously reported [11] suggests that KI is a degraded form of KII. It is therefore likely that a single holoenzyme is present in exocrine cells. In addition no protein kinase, specifically stimulated by cyclic GMP, has been detected in any fraction obtained in the course of purification of the cyclic AMP-dependent protein kinase. A faster and more efficient method than the one previously described [11] allows the purification (5000 times) of the protein kinase catalytic subunit. Analysis of the subunit by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates a molecular weight of 40 000 +/- 1 000. The enzyme phosphorylates specifically histone H2B (Vm = 236 min(-1), Km = 1.15 10(-5) M) and to a lesser extent H2A, H5 and H1 (Vm = 55--77 min(-1), Km 5--25 10(-5) M). Histones H3 and H4 are not phosphorylated. The effect of the heat stable inhibitor, extracted from rat pancreas, on the phosphorylation of H2B has been investigated. The inhibition is of the non competitive type with respect to ATP. The inhibition at various histone concentrations cannot be described by the Michaelis-Menten equation.


[In vitro comparative metabolism of 6,7-3H estrone and 6,7-3H estrone-3-sulfate in maternal and fetal guinea-pig liver].

The metabolism of [6,7-3H] estrone and of [6,7(3)H] estrone-3-sulfate have been comparatively studied in the maternal and fetal guinea-pig livers. The appearance of estradiol-17 beta resulting from the activity of the 17 beta-hydroxysteroid-dehydrogenase is more important in the fetal than in the maternal hepatic tissue. This suggests the direct transformation of estrone-3-sulfate into estradio-3-sulfate in the fetus. After incubation of the [3H] estrone, there is an abundant hepatic conjugation. The glycuroconjugated components are predominant, as well in the maternal as in the fetal hepatic tissue. For the latter-one the sulfoconjugation is inexistant. The sulfatasic activity shown after the incubation of [3H] estrone-3-sulfate is very low in the fetal hepatic tissue; in contrast, this activity is higher in the maternal tissue.


Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence.

Direct spectroscopic measurements of rotational motions of proteins and large protein segments are crucial to understanding the molecular dynamics of protein function. Fluorescent probes and spin labels attached to proteins have proved to be powerful tools in the study of large-scale protein motions. Fluorescence depolarization and conventional electron paramagnetic resonance (EPR) are applicable to the study of rotational motions in the nanosecond-to-microsecond time range, and have been used to demonstrate segmental flexibility in an antibody and in myosin. Very slow rotational motions, occurring in the microsecond-to-millisecond time range, are particularly important in supramolecular assemblies, where protein motions are restricted by association with other molecules. Saturation transfer spectroscopy (ST-EPR), a recently developed electron paramagnetic resonance (EPR) technique that permits the detection of rotational correlation times as long as 1 ms, has been used to detect large-scale rotational motions of spin-labeled proteins in muscle filaments and in membranes, providing valuable insights into energy transduction mechanisms in these assemblies.


Spin label detection of intermolecular interactions in carbonmonoxy sickle hemoglobin.

With recently developed spin label techniques for monitoring macromolecular rotational motion, heme-liganded sickle cell hemoglobin in the presence of inositol hexaphosphate is shown to exhibit restricted motional freedom as compared to liganded normal adult human hemoglobin. This motional restriction is dependent on both hemoglobin concentration and temperature.


[Effect of potassium iodide and perchlorate on the process of thyroid hormone secretion].

The influence of potassium iodide and perchlorate on the parameters characterizing the thypoid hormones secretion, such as the cAMP level in the gland tissue and the number of intracellular colloid droplets under condition of stimulation by thyrotropic hormone was studied. It was shown that the abovementioned parameters were depressed by an excess of iodide, but perchlorate administration prevented the inhibitory effect of iodide. The results obtained favour the conception on the sensitivity of the thyroid adenylate cyclase system to the organic iodine concentration. Apparently and excess of iodide depressed the capacity of perchlorate to influence its concentration in the gland, and thereby the process of iodine organification and of the thyroid hormone secretion maintained at the optimal leve.


[Effect of gangliosides on Na-K-ATPase activity and the conformation of microsomal membranes].

The effect of gangliosides on the activity of Na, K-ATPase and K-dependent nitrophenyl-phosphatase was studied. Low concentrations of gangliosides proved to activate Na, K-ATPase, and high ones to inhibit it. All the concentrations of gangliosides produced only an inhibitory effect on the K-dependent nitrophenyl-phosphatase. Inhibition of the enzyme by gangliosides is reversible and competitive in respect to K+. Calculation of Hill's coefficient demonstrated that both in case of the activating and of the inhibitory action gangliosides acted allosteric effectors. To ascertain the action mechanism of gangliosides on the enzyme their effect on the microsomal membranes was studied by the fluorescent probe method. Gangliosides were found to produce marked conformation shifts in the membranes of the brain microsomes.


[Alterations of Rous virus and polyoma virus during their synthesis in the cells of unnatural hosts].

The line of human embryo fibroblasts transformed by Rous sarcoma virus (strain Schmidt-Ruppin) contained and produced Rous sarcoma virus; this was shown by the complement fixation test, immunofluorescent test, electron microscopy and labelling with 3H-uridine peak in sucrose gradient. Biological properties of the new synthesized virus differed from that of the parent Schmidt-Ruppin strain; the range of the sensitive cells and the protein envelope antigens altered in particular. Analogous results by the change of the viral biological properties produced in the unnatural host tissue were obtained for polioma virus synthesized in the human embryo fibroblasts transformed by this virus.


Direct characterization of beta-adrenoceptors in membranes of immature red blood cells from rats.

By means of the radioactive antagonist ligand (3H)(-) dihydroalprenolol (DHAP) specific binding sites were identified in membrane preparations from red blood cells from rats. These specific sites were characterized as beta-adrenoceptors because of the following reasons: Specific binding of DHAP (in contrast to unspecific binding) was dependent on temperature and time of incubation. Furthermore, specific binding of DHAP showed saturability, temperature-dependent reversibility and high affinity (KD-value of DHAP = 6.51 nM). Specific binding of DHAP was competitively inhibited by beta-adrenergic antagonists (pindolol greater than alprenolol greater than or equal to propranolol greater than practolol) and agonists (isoprenaline greater than adrenaline). The (-) enantiomers of pindolol and isoprenaline showed pronounced higher affinities for the receptor sites than the respective (+) enantiomers. The receptor density in the membrane preparations (pmoles/mg protein) was strongly dependent on the degree of reticulocytosis: The Bmax-values increased more than 4 to 5 fold without alteration of the respective KD-values when reticulocyte counts were enhanced from 3 to 80% treatment of the animals with increasing doses of acetyl phenylhydrazine.


A comparison of myelopathy associated with megavoltage irradiation and remote cancer.

Two middle-aged women with malignancy of the aerodigestive tract were treated by megavoltage irradiation. Both subsequently developed transverse myelopathy. The diagnosis of radiation myelitis was rendered in one. The other was thought to have metastatic compression of the spinal cord; in this case, evidence was such that radiation myelopathy also was appropriate. The irradiation dose-time relationships in both patients were biologically low compared to published isoeffect curves available in the literature. The first patient had injury within the irradiated region and the main neurological damage was found there. In the second patient the entire spinal cord was necrotic, clearly placing the second case outside the radiation myelopathy syndrome. Gross and microscopic examinations of the tissues of these two patients show that the remote effects of malignancy upon the central nervous system can imitate the clinical picture of radiation myelitis.


Evaluation of follow-up services for elderly people prescribed hearing aids: report of a pilot project.

The first stage of a programme to determine the effectiveness of different types of follow-up service for elderly people prescribed hearing aids is described. Follow-up sessions of two hours each were held at fortnightly intervals and were given to groups comprising six patients. Three treatments were compared, one consisting of two follow-up sessions, the second of four sessions, and the third none (the control group). Two groups received each treatment. The results failed to demonstrate any advantage from the treatments. The men, who were more handicapped by their hearing loss, did better than women and there was a predominance of men in the control groups. The necessity of some follow-up service was demonstrated since a substantial proportion of patients were not utilising their aids satisfactorily at six months after issue and had problems which were then resolved. Any follow-up programme must concentrate very heavily on basic handling and maintenance of the aid, for most of the difficulties experienced were with these.


[The mechanism of gonadal differentiation].

The steroid-hormone dependent sexual development presupposes the already sex-specific differentiated gonads. The H-Y antigen is held responsible for the primary differentiation of the indifferent gonadal anlage towards testicular organization. Studies on the function of H-Y antigen, using gonadal cells in vitro, have shown that in the presence of H-Y antigen the gonad differentiates into testicular, in its absence into ovarian structures, independent of whether the sex chromosomes are XX or XY. The gonads of both sexes are endowed with a specific H-Y receptor, other tissues are not. The bipotency of the gonadal anlage arises from this character. Some disorders of sexual development presumably go back to mutations affecting the genetic control of the H-Y gene and the function of H-Y antigen, respectively. It is discussed that the Y chromosome may have a mere regulatory function and not include the H-Y gene. Thus, the primary differentiation of the gonad should be dependent exclusively on whether or not the H-Y gene is active.


Modulation of noradrenergic transmission in the rabbit ear artery by dopamine.

1. The effects of dopamine on vasoconstrictor responses to field stimulation of sympathetic nerves and to exogenous noradrenaline were studied in the isolated ear artery of the rabbit. Responses to noradrenaline were unchanged at the start of the dopamine infusions but were enhanced as the infusions continued and also after cessation of the infustion. 2 Dopamine (0.5 muM) reduced the stimulation-induced efflux of tritium from segments of ear artery labelled with [3H]-noradrenaline. The reduction persisted during 65 min of dopamine infusion, after which time the vasoconstrictor responses had generally recovered to 93% of control level. On ceasing the infusion, the stimulation-induced efflux and the vasoconstrictor responses were enhanced. 3 Metoclopramide, haloperidol and ergometrine, each in a concentration of 0.2 muM, prevented the inhibitory effect of 0.5 muM dopamine on the stimulation-induced tritium release, but not the inhibitory effect of 0.5 muM noradrenaline. Phenoxybenzamine (0.2 and 1 muM) and phentolamine (1 muM) prevented the inhibitory effects of both noradrenaline and dopamine on the stimulation-induced efflux, and phentolamine (0.2 muM) prevented the inhibition of the stimulation-induced release by noradrenaline but only partially prevented the inhibitory effect of dopamine on the stimulation-induced efflux. 4 A possible role for dopamine in the modulation of noradrenergic transmission is suggested.