bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of mitochondrial functions by glucagon treatment. Evidence that effects are not artifacts of mitochondrial isolation
Authors: Jensen C B , Sistare F D , Hamman H C , Haynes R C
Published: August 2015

(1) Activation of rat liver mitochondrial functions following glucagon treatment was demonstrated in mitochondria that had not been isolated by the conventional technique of differential centrifugation and washing in sucrose solutions. Crude liver homogenates in 0.3 M-sucrose or 0.15 M-KCl prepared from rats treated with glucagon showed stimulation of State-3 and uncoupled respiration, carboxylation of pyruvate, and citrulline synthesis comparable with those previously reported in isolated mitochondria. (2) During the isolation procedure of mitochondria the hormonal stimulations of pyruvate carboxylation and citrulline formation were shown not to be enhanced by sequential washing. (3) Mitochondria isolated from glucagon-treated rats by differential centrifugation and washing in 0.3 M-mannitol/1 mM-EGTA, pH 7.0, exhibited a mean rate of citrulline synthesis that was greater than twice that of the control. Liver homogenates prepared in 0.3 M-sucrose or 0.3 M-mannitol showed identical rates of State-3 respiration and percentage stimulations of respiration by glucagon treatment. (4) Addition of glucagon led to a rapid accumulation of malate and aspartate and decreased the amounts of glutamate and citrate in isolated hepatocytes incubated with L-lactate. When gluconeogenesis was inhibited at the phosphoenolpyruvate carboxykinase (EC 4.1.1.32) reaction these phenomena were accentuated, lending support to the interpretation that they are the direct result of stimulation of carboxylation and oxidation reactions in the mitochondria. These results do not support the proposal [Siess, Fahimi & Wieland (1981) Hoppe-Seyler's Z. Physiol. Chem. 362. 1643-1651] that the mitochondrial effects of glucagon treatment result from a stabilization of mitochondria to detrimental effects of sucrose during their isolation. (5) The mean hormonal stimulation of pyruvate carboxylation in mitochondria isolated in 0.3 M-sucrose was shown to be approx. 2.5-fold when assayed either at 37 degrees C or 25 degrees C. In contrast, on the basis of similar experiments, Siess et al. (1981) concluded that the effects of glucagon on hepatic mitochondria are not characteristic of a true hormonal stimulation. Our data indicate this conclusion to be unjustified.
Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects
Authors: Malaisse W J , Sener A , Welsh M , Malaisse-Lagae F , Hellerström C , Christophe J
Published: August 2015

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as ‘partners’ in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.
Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells
Authors: Kondo M , Koshihara Y , Kawamura M , Murota S
Published: August 2015

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.
Hormonal activation of type-L hormone-sensitive lipase measured in defatted heart powders
Authors: Palmer W K , Kane T A
Published: August 2015

Adrenaline, 3-isobutyl-1-methylxanthine (MIX) and dibutyryl cyclic AMP (Bt2 cyclic AMP) stimulated type-L hormone-sensitive lipase (HSL) activity when measurements were made on defatted rat heart powders. These lipolytic agents stimulated the activity of this enzyme in a time- and dose-dependent manner. This activation was reversible, because removal of adrenaline from the perfusate was accompanied by the return of type-L HSL activity to control levels. We have reported [Palmer, Caruso & Oscai (1981) Biochem. J. 198, 159-166] that perfusion with low levels of adrenaline, MIX or Bt2 cyclic AMP reduced type-L HSL activity below control levels when measurements were made in aqueous homogenates. However, in the present study, when activities were measured in acetone/diethyl ether heart powders, all concentrations of these agents studied stimulated enzyme activity, and at no concentration was there enzyme inhibition. These data suggest that acetone/diethyl ether treatment may remove a factor that plays a role in type-L HSL regulation. Type-L HSL activity measured in acetone/diethyl ether powders of control and stimulated rat heart exhibited properties that include alkaline pH optimum, serum requirement, activation by heparin and inhibition by high salt and protamine sulphate. These characteristics, in addition to the stability of the enzyme to treatment with organic solvents, fulfil the requirements for the type-L HSL classification.
Sensitivity to insulin of glycolysis and glycogen synthesis of isolated soleus-muscle strips from sedentary, exercised and exercise-trained rats
Authors: Espinal J , Dohm G L , Newsholme E A
Published: August 2015

The half-maximal stimulation of the rates of glycolysis and glycogen synthesis in soleus-muscle strips from sedentary animals occurred at a concentration of insulin of about 100 microunits/ml. In soleus-muscle strips from exercise-trained rats (5 weeks of treadmill training), half-maximal stimulation of the rate of glycolysis occurred at about 10 microunits of insulin/ml, whereas that for glycogen synthesis occurred between 10 and 100 microunits of insulin/ml. The sensitivity of glycolysis to insulin after exercise training is similar to that of adipose tissue from sedentary animals. This finding suggests that, in sedentary animals, the effects of normal changes in insulin concentration may affect muscle primarily indirectly via the anti-lipolytic effect on adipose tissue, whereas after training insulin may effect the rate of glycolysis in muscle directly. A single period of exercise did not change the sensitivity of glycolysis in soleus muscle to insulin, nor probably that of glycogen synthesis. It is suggested that the improvement in insulin sensitivity of glycolysis in muscle caused by exercise-training could account, in part, for the well-established improvement in glucose tolerance and insulin sensitivity observed in man and rats after exercise-training.
Histamine H2 receptors on foetal-bovine articular chondrocytes
Authors: Taylor D J , Yoffe J R , Woolley D E
Published: August 2015

The dose-response curve of histamine-induced cyclic AMP elevation in monolayer cultures of primary foetal-bovine articular chondrocytes was displaced to the right by cimetidine. In addition, H2 but not H1 antagonists prevented the histamine-induced cyclic AMP elevation, suggesting histamine activates chondrocyte adenylate cyclase through an H2 receptor.
Bovine serum albumin decreases 4-methyl-2-oxovalerate utilization by isolated rat hepatocytes
Authors: Livesey G
Published: August 2015

Binding of 4-methyl-2-oxo[1-14C]valerate to defatted bovine serum albumin inhibited the utilization of this 2-oxo acid by fed-rat hepatocytes in vitro. With 0-50g of albumin/l in the presence of 0.05mM 2-oxo acid or on increasing the 2-oxo acid concentration from 0 to 2mM in the presence of 26g of albumin/l, the extent of inhibition was essentially dependent on the change in the free 2-oxo acid concentration. Intrahepatocyte 4-methyl-2-oxo[1-14C]valerate concentrations were similar to extracellular free 2-oxo acid concentrations, suggesting equilibration so that the plasma membrane appears not to be rate-limiting for the utilization of this substrate by the isolated liver cells.
Changes in the cytochrome composition of Rhodopseudomonas sphaeroides grown aerobically, photosynthetically and on dimethyl sulphoxide
Authors: Ward J A , Hunter C N , Jones O T G
Published: August 2015

Several strains and mutants of Rhodopseudomonas sphaeroides can be grown anaerobically in the dark in the presence of dimethyl sulphoxide as an electron acceptor. During adaptation to this fermentative mode of growth, two major c-type cytochromes are synthesized, one with Mr 45 000 and the second with Mr 20 000 and a midpoint potential of +120 mV. These cytochromes are barely detectable in membranes prepared from cells grown in aerobic or photosynthetic conditions. An electrophoretic method is presented for the detection of the b-type and c-type cytochromes of pigmented or unpigmented membranes. The method resolves three b-type cytochromes and four c-type cytochromes in membranes from aerobically and photosynthetically grown cells.
Isolation and renaturation of α2-macroglobulin receptor from diploid human fibroblasts
Authors: Frey J , Afting E G
Published: August 2015

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.
Influence of phospholipids on the activity of phosphate-dependent glutaminase in extracts of rat liver mitochondria
Authors: McGivan J D , Bradford N M
Published: August 2015

Liver glutaminase can be solubilized from frozen-and-thawed mitochondria by treatment with phospholipase A2. Solubilization by this technique markedly changes the kinetic properties of the enzyme. The properties of the membrane-bound form of the enzyme are partially restored by adding phosphatidylcholine or phosphatidylethanolamine to the phospholipase extract. It is concluded that the kinetic properties of liver glutaminase are a function of the interaction of this enzyme with membrane phospholipids.
Preparation of a low-density species of endocytic vesicle containing immunoglobulin A
Authors: Mullock B M , Luzio J P , Hinton R H
Published: August 2015

Immunoglobulin A is transported across hepatocytes in specialized vesicles. A population of endocytic vesicles of approx. 140 nm diameter, containing immunoglobulin A, has now been separated from all other major cytoplasmic organelles, including plasma membrane and lysosomes, by sequential centrifugation on Ficoll/sucrose and Metrizamide gradients.
Reversible alteration of hepatic messenger RNA species for peroxisomal and non-peroxisomal proteins induced by the hypolipidaemic drug Wy-14,643
Authors: Chatterjee B , Demyan W F , Lalwani N D , Reddy J K , Roy A K
Published: August 2015

Extensive peroxisomal proliferation in the hepatic parenchymal cells was observed when male rats were given a diet containing 0.1% Wy-14,643 [(4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a potent lipid-decreasing drug. This drug also caused a marked increase in the concentrations of the mRNA species coding for four proteins with Mr 77000, 61000, 43000 and 31000, and a similar decrease in the concentrations of three mRNA species coding for proteins of Mr 25000, 24000 and 19000. Specific immunoprecipitation studies identified the proteins of Mr 19000, 43000 and 77000 as alpha 2u-globulin, 3-ketoacyl-CoA thiolase (EC 2.3.1.16) and enoyl-CoA hydratase (EC 4.2.1.17) respectively. Comparisons of the Mr values suggest that the 61000- and 31000-Mr proteins may be equivalent to two additional peroxisomal enzymes, namely catalase (Mr 61000) and uricase (Mr 31000). The identity of the mRNA species coding for the 25000- and 24000-Mr proteins is at present unknown.
An assessment of some methodological criticisms of studies of RNA efflux from isolated nuclei
Authors: Agutter P S
Published: August 2015

RNA efflux from isolated nuclei can be studied either as a means of elucidating the general mechanism of nucleo-cytoplasmic RNA transport, or as part of an investigation of the processing and utilization of particular gene transcripts. The present paper describes an assessment of three methodological criticisms of RNA-efflux measurements that are made for the former reason: for such measurements, it is sufficient to show that the post-incubation supernatant RNA is similar overall to homologous cytoplasmic mRNA, rather than to nuclear RNA, that is nevertheless of intranuclear origin, and that alterations to the medium during experiments do not markedly perturb this general nuclear restriction. The results seem to justify the following conclusions. (1) Although degradation of the nuclear RNA occurs during incubation in vitro, this process does not account for the appearance of RNA in the postnuclear supernatant. The degradation can be largely prevented by the addition of serine-proteinase inhibitors without altering the RNA efflux rate. (2) Some adsorption of labelled cytoplasmic RNA to the nuclear surface occurs during both isolation and incubation of the nuclei, and some desorption occurs during incubation. However, these effects introduce errors of less than 10% into the measurements of efflux rates. (3) Exogenous acidic polymers, including polyribonucleotides, disrupt nuclei and increase the apparent RNA efflux rate by causing leakage of nuclear contents. However, this effect can largely be overcome by including the nuclear stabilizers spermidine, Ca2+ and Mn2+ in the medium. In terms of this assessment, it appears that RNA efflux from isolated nuclei in media containing nuclear stabilizers serves as a reasonable model for transport in vivo.
Inhibition of cell division by interferons. The relationship between changes in utilization of thymidine for DNA synthesis and control of proliferation in Daudi cells
Authors: Gewert D R , Moore G , Clemens M J
Published: August 2015

Inhibition of the proliferation of Daudi cells by exposure to human lymphoblastoid interferons is associated with an early and marked decrease in the incorporation into DNA of exogenous [3H]thymidine when cells are incubated with trace amounts of this precursor. In contrast, incorporation of exogenous deoxyadenosine into DNA is unchanged under the same conditions. Interferon treatment results in a lowering of thymidine kinase activity, an effect which may be largely responsible for the inhibition of incorporation of labelled thymidine into DNA. At higher concentrations of exogenous thymidine, which minimize the contribution of intracellular sources to the dTTP pool, the inhibition of thymidine incorporation is abolished. Under conditions in which exogenous thymidine is rigorously excluded from the medium or, conversely, in which cells are entirely dependent on exogenous thymidine for growth, the magnitude of the inhibition of cell proliferation by interferons is the same as under normal culture conditions. We conclude that, even though cell growth is impaired, the rate of DNA synthesis is not grossly inhibited up to 48 h after commencement of interferon treatment. Furthermore, changes in neither the utilization of exogenous thymidine nor the synthesis of nucleotides de novo are responsible for the effect on cell proliferation.
Resistance to fusidic acid in Escherichia coli mediated by the type I variant of chloramphenicol acetyltransferase. A plasmid-encoded mechanism involving antibiotic binding
Authors: Bennett A D , Shaw W V
Published: August 2015

Plasmid-encoded fusidic acid resistance in Escherichia coli is mediated by a common variant of chloramphenicol acetyltransferase (EC 2.3.1.28), an enzyme which is an effector of chloramphenicol resistance. Resistance to chloramphenicol is a consequence of acetylation of the antibiotic catalysed by the enzyme and the failure of the 3-acetoxy product to bind to bacterial ribosomes. Cell-free coupled transcription and translation studies are in agreement with genetic studies which indicated that the entire structural gene for the type I chloramphenicol acetyltransferase is necessary for the fusidic acid resistance phenotype. The mechanism of resistance does not involve covalent modification of the antibiotic. The other naturally occurring enterobacterial chloramphenicol acetyltransferase variants (types II and III) do not cause fusidic acid resistance. Steady-state kinetic studies with the type I enzyme have shown that the binding of fusidic acid is competitive with respect to chloramphenicol. The inhibition of polypeptide chain elongation in vitro which is observed in the presence of fusidic acid is relieved by addition of purified chloramphenicol acetyltransferase, and equilibrium dialysis experiments with [3H]fusidate and the type I enzyme have defined the stoichiometry and apparent affinity of fusidate for the type I enzyme. Further binding studies with fusidate analogues, including bile salts, have shown some of the structural constraints on the steroidal skeleton of the ligand which are necessary for binding to the enzyme. Determinations of antibiotic resistance levels and estimates of intracellular chloramphenicol acetyltransferase concentrations in vivo support the data from experiments in vitro to give a coherent mechanism for fusidic acid resistance based on reversible binding of the antibiotic to the enzyme.
Complex-formation and inhibition of urokinase by blood plasma proteins
Authors: Waller E K , Schleuning W D , Reich E
Published: August 2015

We have studied the formation of covalent complexes between 125I-urokinase (125I-UK) and proteins in human plasma. Although 125I-UK reacts with many proteinase inhibitors in purified systems, the predominant complexes formed in plasma are with antithrombin III (ATIII) and alpha 2-macroglobulin (alpha 2M). 125I-UK interacts with purified alpha 2M or alpha 2M in plasma to form a characteristic pattern of multiple complexes whose Mr values by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis are in the range of 380 000-720 000, under non-reducing conditions, and 180 000-430 000 after reduction. We also examined the inhibition of UK amidolytic activity by plasma and by purified ATIII. In the presence of saturating concentrations of ATIII and heparin, an apparent first-order rate constant of 6.8 X 10(-1) s-1 was calculated for the inhibition of urokinase. In contrast, the rate constant for the formation of covalent ATIII-UK complexes was lower, suggesting the inhibition of UK proceeds first via the formation of transient non-covalent intermediates that are then transformed more slowly into covalent end products. The observed rate constants for enzyme inhibition or complex-formation with plasma or purified inhibitors are insufficient to account for the reported clearance rate of injected UK in vivo.
Monoclonal antibody against the N-terminal end of human plasma fibronectin
Authors: Vartio T , Salonen E M , De Petro G , Barlati S , Miggiano V , Stähli C , Virgallita G , Takács B , Vaheri A
Published: August 2015

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.
Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria
Authors: Bauché F , Sabourault D , Giudicelli Y , Nordmann J , Nordmann R
Published: August 2015

In rat liver hypo-osmotically treated mitochondria, 2-mercaptoacetate inhibits respiration induced by palmitoyl-CoA, octanoate or butyryl-CoA only when the reaction medium is supplemented with ATP. Under this condition, NADH-stimulated respiration is not affected. In liver mitochondrial matrix, the presence of ATP is also required to observe a 2-mercaptoacetate-induced inhibition of acyl-CoA dehydrogenases tested with palmitoyl-CoA, butyryl-CoA or isovaleryl-CoA as substrate. As the oxidation of these substrates is also inhibited by the incubation medium resulting from the reaction of 2-mercaptoacetate with acetyl-CoA synthase, with conditions under which 2-mercaptoacetate has no effect, 2-mercaptoacetyl-CoA seems to be the likely inhibitory metabolite responsible for the effects of 2-mercaptoacetate. Kinetic experiments show that the main effect of the 2-mercaptoacetate-active metabolite is to decrease the affinities of fatty acyl-CoA dehydrogenases towards palmitoyl-CoA or butyryl-CoA and of isovaleryl-CoA dehydrogenase towards isovaleryl-CoA. Addition of N-ethylmaleimide to mitochondrial matrix pre-exposed to 2-mercaptoacetate results in the immediate reversion of the inhibitions of palmitoyl-CoA and isovaleryl-CoA dehydrogenations and in a delayed reversion of butyryl-CoA dehydrogenation. These results led us to conclude that (i) the ATP-dependent conversion of 2-mercaptoacetate into an inhibitory metabolite takes place in the liver mitochondrial matrix and (ii) the three fatty acyl-CoA dehydrogenases and isovaleryl-CoA dehydrogenase are mainly competitively inhibited by this compound. Finally, the present study also suggests that the inhibitory metabolite of 2-mercaptoacetate may bind non-specifically to, or induce conformational changes at, the acyl-CoA binding sites of these dehydrogenases.
The storage products in genetic and swainsonine-induced human mannosidosis
Authors: Cenci di Bello I , Dorling P , Winchester B
Published: August 2015

Swainsonine induces the accumulation of mannose-rich oligosaccharides in human fibroblasts. The composition of the storage products shows that swainsonine completely inhibits lysosomal alpha-D-mannosidase and alters processing of glycoproteins by inhibiting Golgi alpha-D-mannosidase II. Comparison of the storage products in genetic and swainsonine-induced mannosidosis suggests that human fibroblasts contain a lysosomal alpha-D-mannosidase that is unaffected in genetic mannosidosis.
Bidirectional transport of glutamine across the cell membrane in rat liver
Authors: Fafournoux P , Demigné C , Rémésy C , Le Cam A
Published: August 2015

Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.
Concomitant increase in hepatic triacylglycerol biosynthesis and cytosolic fatty-acid-binding-protein content after feeding rats with a cholestyramine-containing diet
Authors: Kempen H J M , Glatz J F C , de Lange J , Veerkamp J H
Published: August 2015

Cholestyramine feeding of rats increased the rate of palmitate and glycerol incorporation into triacylglycerols of isolated hepatocytes. Concomitantly an increase of fatty-acid binding by hepatic cytosolic proteins was observed, which could be attributed to an elevation of the content of the fatty-acid-binding protein (Mr 12000). The involvement of this protein in cholesterol, bile-acid and triacylglycerol metabolism is discussed.
Two different protein kinase activities are associated with the insulin receptor
Authors: Gazzano H , Kowalski A , Fehlmann M , Van Obberghen E
Published: August 2015

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.
The role of insulin, corticosterone and other factors in the acute recovery of muscle protein synthesis on refeeding food-deprived rats
Authors: Millward D J , Odedra B , Bates P C
Published: August 2015

Measurements of changes in muscle protein synthesis, insulin and corticosterone in vivo in refed food-deprived rats, some after pretreatment with anti-insulin serum or corticosterone, indicate that the acute increase in protein synthesis (20-40 min) requires (a) insulin, (b) a fall in corticosterone, since corticosterone acts at least in part by blocking insulin action, and (c) at least one other independent anabolic factor.
Metabolism of rat bone proteoglycans in vivo
Authors: Prince C W , Rahemtulla F , Butler W T
Published: August 2015

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.
The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions
Authors: Owen R W , Bilton R F
Published: August 2015

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.
Glycosylation of proteins from sugar nucleotides by whole cells. Effect of ammonium chloride treatment on mouse thymocytes
Authors: Cecchelli R , Cacan R , Hoflack B , Verbert A
Published: August 2015

When thymocytes are treated with iso-osmotic NH4Cl, the sugar incorporation into endogenous acceptors from labelled sugar nucleotides is largely increased compared with that in control thymocytes. This effect was obtained with labelled GDP-mannose, UDP-galactose and CMP-N-acetylneuraminic acid. The stimulation observed with NH4Cl-treated thymocytes does not involve the glycosylation of exogenous acceptors, and it was proved that the NH4Cl treatment (1) does not stimulate glycosyltransferase activities themselves, (2) does not lead to the release of soluble glycosyltransferases as the result of an extensive lysis of the thymocytes and (3) does not cause the emergence of glycosyltransferases at the cell surface. In fact, electron-microscopy observations showed that, although marked changes had occurred in the cytoplasm, the plasma membrane is sufficiently maintained to allow the cell to keep roughly its original shape and to retain the intracellular vesicles. We thus demonstrate that this stimulation is due to an enhancement of the entry of sugar nucleotides into the cell. As demonstrated by the inclusion of Trypan Blue within the cells, and the non-stimulation of glycosylation of exogenous large-molecular-mass acceptors, the effect of NH4Cl seems to be limited to the penetration of small-molecular-sized compounds through the plasma membrane. Thus NH4Cl treatment allows the labelled sugar nucleotides to penetrate the cell and to behave as the cellular pool to be utilized for glycosylation by intracellular vesicles.
Regulation of lipogenesis in vivo by glucose availability and insulin secretion in maternal and foetal tissues during late gestation in the rat. Effect of glucose intubation, streptozotocin-induced diabetes and starvation
Authors: Lorenzo M , Benito M , Caldés T , Medina J M
Published: August 2015

Administration of an oral load of glucose did not change the rate of lipogenesis in maternal liver during late gestation. However, streptozotocin-induced diabetes or starvation decreased maternal liver lipogenesis at 20-22 days of gestation. Glucose intubation, on the other hand, increased foetal lipogenesis at 21-22 days. In addition, maternal starvation decreased foetal lipogenesis and plasma insulin concentration. However, chronic hyperglycaemia induced by streptozotocin administration to the mother did not change foetal liver lipogenesis.
Purification and properties of glutathione S-transferases from larvae of Wiseana cervinata
Authors: Clark A G , Drake B
Published: August 2015

The glutathione S-transferases from the porina moth, Wiseanna cervinata, were purified by affinity chromatography, cation-exchange chromatography and preparative isoelectrofocusing. The major transferase (IV) was purified to homogeneity by a factor of 530-fold with a yield of 83%. Other transferases present were purified to a smaller degree (approx. 50-fold) to a stage of near-homogeneity. The transferases examined all had Mr values about 45 000-50 000. They appeared to be homodimers of either of two types of subunit, of Mr 22 800 and 24 600. Enzymes consisting of the different types of subunit were not immunologically cross-reactive. The major enzyme fractions separated by cation-exchange chromatography were both active with 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and iodomethane, but were inactive with 4-nitropyridine N-oxide, 1,2-epoxy-3-(p-nitrophenoxy)propane, bromosulphophthalein and p-nitrobenzyl chloride. The kinetics of the enzyme-catalysed reaction with enzyme IV were non-Michaelean with respect to both substrates. Both products were inhibitory. The results appear to be compatible with a random steady-state mechanism. It is concluded that these enzymes are very similar, in their physical and chemical constitution, in their catalytic properties and in their relationships with each other, to those enzymes that have been isolated from vertebrate organisms.
Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes
Authors: Pohlmann R , Krüger S , Hasilik A , von Figura K
Published: August 2015

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.
Cystatin. Amino acid sequence and possible secondary structure
Authors: Schwabe C , Anastasi A , Crow H , McDonald J K , Barrett A J
Published: August 2015

The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure.
Redox potentials of algal and cyanobacterial flavodoxins
Authors: Sykes G A , Rogers L J
Published: August 2015

The redox potentials of flavodoxins from the cyanobacteria Synechococcus PCC 6301 (formerly Anacystis nidulans) and Nostoc strain MAC, and from the red alga Chondrus crispus, were determined by potentiometric titration. For the oxidized-semiquinone interconversion the potentials at pH 7.0 of the three flavodoxins were between −210 and −235 mV, and these were pH-dependent over the range pH 6.9-8.2. For the semiquinone-reduced interconversion the potentials of the cyanobacterial flavodoxins were close to −414 mV, and that for the algal flavodoxin, −370 mV, is the highest reported in this group of flavoproteins.
Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion
Authors: Lang R D A , Wickenden C , Wynne J , Lucy J A
Published: August 2015

Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.
Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240000-Mr and 116000-Mr polypeptides in the presence of a bifunctional thiol probe
Authors: Harris W E , Stahl W L
Published: August 2015

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4′-difluoro-3,3′-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.
Effect of thioacetamide on cytochrome P-450 synthesis in rat liver
Authors: Satyabhama S , Padmanaban G
Published: August 2015

A single injection of thioacetamide to rats results in a striking decrease in liver cytochrome P-450. The drug blocks the phenobarbitone-mediated induction of cytochrome P-450b. Under these conditions, there is a decrease in the synthesis of cytochrome P-450b, which is essentially due to a decrease in the amount of translatable mRNA for cytochrome P-450b. This effect of thioacetamide is not due to a general effect on protein synthesis or polyribosome stability. Multiple injections of thioacetamide also decrease the synthesis of cytochrome P-450b and the amounts of its translatable mRNA.
Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda
Authors: Rytka J , Bilinski T , Labbe-Bois R
Published: August 2015

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type. The excreted porphyrins comprised mainly dehydroisocoproporphyrin. Analysis of uroporphyrinogen decarboxylase activity in the cell-free extract revealed a 70-80% decrease of activity in the mutant and showed that the relative rates of the different decarboxylation steps were modified with the mutant enzyme. A 2-3-fold increase in 5-aminolaevulinate synthase activity was measured in the mutant. The biochemical characteristics of the Sm1 mutant are very similar to those described for porphyria cutanea tarda.
Enzymic characteristics of the isoenzymes of rat epididymal neutral α-mannosidases and their changes during development in vivo
Authors: Dutta P , Majumder G C
Published: August 2015

Three isoenzymic species of neutral alpha-mannosidase (I, II and III) were partially purified from rat epididymis and characterized. Calcium phosphate gel preferentially adsorbed acidic alpha-mannosidase activity, thereby removing the acidic enzyme from the neutral mannosidases. The neutral enzymes, which are of cytosolic origin, require Co2+ for activity, and Mn2+ can substitute partially for Co2+. Mg2+ or Ca2+ had little effect on the activity of the isoenzymes, whereas Zn2+ (100 microM) was a potent inhibitor of the mannosidases. The Km values of mannosidases I, II and III for Co2+ were 10, 10 and 2.7 mM respectively. There was marked alteration of the specific activity of the neutral alpha-mannosidases during epididymal development in vivo. The specific activities of mannosidases I and II were relatively high in the young (24 days) rats, and during the subsequent development the specific activities decreased markedly (approx. 2-3-fold). On the contrary, mannosidase III, which showed relatively low specific activity during 24-45 days of age, increased markedly (approx. 2-fold) as the animals reached adulthood.
Characterization of the C1q receptor on a human macrophage cell line, U937
Authors: Arvieux J , Reboul A , Bensa J C , Colomb M G
Published: August 2015

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.
Antizyme to ornithine decarboxylase is present in the liver of starved rats
Authors: Fujita K , Matsufuji S , Murakami Y , Hayashi S
Published: August 2015

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.