bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of lysophospholipids in intact rat islets. The insulin secretagogue p-hydroxymercuribenzoic acid impairs lysophosphatidylcholine catabolism and permits its accumulation
Authors: Metz S A
Published: August 2015

Although recent studies implicate lysophospholipids (lyso-PLs) in stimulus-secretion coupling in the pancreatic islet, almost no data on lyso-PL metabolism therein exist. Therefore, intact rat islets were loaded with insulinotropic and non-toxic concentrations of 1-[14C]palmitoyl-lysophosphatidylcholine (lyso-PC) via transbilayer movement, and its metabolic fate was studied. The time-dependent hydrolysis of lyso-PC to fatty acid (lysophospholipase activity), its conversion to phosphatidylcholine (putative acyltransferase activity) and, to a lesser degree, the appearance of label in phosphatidylethanolamine (putative transacylase or base exchange activity) were observed. p-Hydroxymercuribenzoic acid (PHMB) at 100 microM (a concentration previously demonstrated to elicit potent exocytotic insulin release) inhibited all three activities (by 56, 46 and 75%, respectively) and led to the intracellular accumulation of lyso-PC. Antimycin A inhibited phosphatidylcholine formation but not lysophospholipase activity; lyso-PC did not accumulate, implying that blockade of both of the major metabolic pathways is required to induce a detectable increment in lyso-PC levels. Calculations derived from data using the lowest effective insulinotropic concentration of lyso-PC suggested that increments in lyso-PC accumulation at critical membrane sites of less than 10-15% above basal values are sufficient to trigger insulin release. Since PHMB elicited increments of 50-100% in lyso-PC after its translocation into islets, support is provided for the earlier contention that lyso-PLs mediate the insulinotropic effect of PHMB. In addition, these studies may provide a more precise experimental paradigm for future studies of islet lyso-PL metabolism.
A novel whey protein synthesized only in late lactation by the mammary gland from the tammar (Macropus eugenii)
Authors: Nicholas K R , Messer M , Elliott C , Maher F , Shaw D C
Published: August 2015

A major whey protein which appears in milk from the tammar wallaby (Macropus eugenii) only during the second half of lactation (late lactation protein-A, LLP-A) was purified to apparent homogeneity by ion-exchange chromatography and gel filtration. An Mr of 21,600 +/- 2000 was calculated from its amino acid composition. A computer-based comparison of the sequence of the first 69 amino acid residues with the Atlas of Protein Sequence data base showed no significant homology with known proteins. Antiserum to LLP-A was prepared in rabbits, and single radial immunodiffusion was used to measure the amounts of LLP-A in milk during the first 40 weeks of lactation. LLP-A was first detected at 26 weeks; thereafter its concentration increased abruptly, to reach a maximum of 26 g/l at approx. 36 weeks of lactation. Explants prepared from mammary gland biopsies at 20 and 35 weeks of lactation were exposed to [3H]amino acids for 8 h; immunoprecipitation of tissue extracts showed that, whereas the rate of casein synthesis was the same at both stages of lactation, LLP-A was synthesized only by the 35-week mammary gland.
Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate
Authors: Shears S B , Storey D J , Morris A J , Cubitt A B , Parry J B , Michell R H , Kirk C J
Published: August 2015

We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.
The effects of chemically modifying serum apolipoproteins on their ability to activate lipoprotein lipase
Authors: Dodds P F , Lopez-Johnston A , Welch V A , Gurr M I
Published: August 2015

Lipoprotein lipase activity was measured in an acetone-dried-powder preparation from rat epididymal adipose tissue using pig serum or pig serum lipoprotein, which had been chemically modified, as activator. Modification of acidic amino acids of lipoproteins with NN-dimethyl-1,3-diamine resulted in a complete loss of ability to activate lipoprotein lipase. Modification of 34% of lipoprotein arginine groups with cyclohexanedione resulted in the loss of 75% of the activation of lipoprotein lipase; approx. 42% of the original activity was recovered after reversal of the modification. This effect was dependent on the cyclohexanedione concentration. Modification of 48% of lipoprotein lysine groups with malonaldehyde decreased the maximum activation by 20%, but three times as much lipoprotein was required to achieve this. Non-enzymic glycosylation of lipoprotein with glucose, under a variety of conditions resulting in up to 28 nmol of glucose/mg of protein, had no effect upon the ability to activate lipoprotein lipase. In contrast non-enzymic sialylation resulted in a time-dependent loss of up to 60% of ability to activate lipoprotein lipase. Reductive methylation and acetoacetylation of serum did not affect the ability to activate lipoprotein lipase. The results are compared to the effects of similar modifications to low density lipoproteins on receptor-mediated endocytosis.
Adipocyte insulin receptor. Generation of a cryptic domain of the α-subunit during internalization of hormone-receptor complexes
Authors: Berhanu P , Saunders D J , Brandenburg D
Published: August 2015

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.
Protein synthesis and degradation in isolated muscle. Effect of ω3 and ω6 fatty acids
Authors: Palmer R M , Wahle K W J
Published: August 2015

The ability of derivatives of the essential fatty acids linoleic acid (C18:2, omega 6) and alpha-linolenic acid (C18:3, omega 3) to stimulate rates of protein synthesis and degradation was investigated in isolated intact muscles from fasted rabbits. Both omega 6 derivatives examined, arachidonic acid (C20:4, omega 6) and dihomo-gamma-linolenic acid (C20:3, omega 6), when added at concentrations up to 1 microM, stimulated the rate of protein synthesis and the release of prostaglandin F2 alpha (PGF2 alpha). Metabolites of the omega 6 series, namely eicosapentaenoic acid (C20:5, omega 3) and docosahexaenoic acid (C22:6, omega 3), were without effect on the rate of protein synthesis and resulted in a decrease in the release of PGF2 alpha. None of the fatty acids had a significant effect on the rate of protein degradation. Although insulin (100 mu units/ml) also stimulated rates of protein synthesis when added alone, none of the omega 3 or omega 6 fatty acids, when added with insulin at concentrations of 0.2 microM, potentiated the effect of the hormone.
Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C
Authors: McDonald J R , Gröschel-Stewart U , Walsh M P
Published: August 2015

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.
The invasive adenylate cyclase of Bordetella pertussis. Intracellular localization and kinetics of penetration into various cells
Authors: Farfel Z , Friedman E , Hanski E
Published: August 2015

The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5′-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane.
The activity of the pyruvate dehydrogenase complex in heart and liver from mice during the development of obesity and insulin resistance
Authors: Caterson I D , Astbury L D , Williams P F , Vanner M A , Cooney G J , Turtle J R
Published: August 2015

The amount of pyruvate dehydrogenase in the active form (PDHa) was increased 1.7-fold compared with controls in heart muscle of mice 1 week after induction of obesity with a single injection of gold-thioglucose. At 4 weeks post injection, the amount of PDHa was decreased to 32% of control, a value which was observed in later stages of the obesity syndrome. In contrast, liver PDHa was increased and remained at an increased activity during the development of obesity. Despite normal post-prandial serum insulin contents, liver membrane insulin-receptor numbers were decreased 1 week after gold-thioglucose injection, and there was no change in receptor affinity. The decrease in heart PDHa in the obese animals was reversed by a single dose of 2-tetradecylglycidic acid, but this inhibitor of mitochondrial fatty acid oxidation did not affect liver PDHa in these animals. These early and diverse changes in PDHa argue for a multifactorial aetiology in the development of the whole-body insulin resistance seen in older gold-thioglucose-treated obese animals.
Gene expression of type I, III and IV collagens in hepatic fibrosis induced by dimethylnitrosamine in the rat
Authors: Ala-Kokko L , Pihlajaniemi T , Myers J C , Kivirikko K I , Savolainen E R
Published: August 2015

Dimethylnitrosamine (DMN)-induced hepatic fibrosis was used as an experimental model to study collagen-gene expression during liver fibrogenesis. Increase in the concentrations of the mRNAs for type I, III, and IV collagens was found to be an early event in the development of hepatic fibrosis, as the mRNAs for all three collagen types showed a definite increasing tendency by day 7 of DMN treatment. Prolyl 4-hydroxylase (EC 1.14.11.2) and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activities were also distinctly elevated at this stage, whereas no increase could be detected in the liver collagen content. The increase in the mRNAs for type I collagen was the smallest and that for type IV collagen the greatest at all the time points studied. The relative concentrations of the mRNAs for the three collagen types on day 21 of DMN treatment were 350% of the control mean for type I collagen, 490% for type III and 660% for type IV. The data further indicate that the proportions of the mRNAs for the three collagen types are 1.0:0.9:0.2 in normal rat liver, 1.0:1.4:0.8 on day 14 of DMN treatment, and 1.0:1.3:0.5 on day 21. The early marked increase in the mRNA for type IV collagen suggests that enhanced production of basement-membrane collagen may be an early event in the development of hepatic fibrosis.
Participation of the lone tryptophan residue of rat α-foetoprotein in its drug-binding sites. Comparison with rat serum albumin
Authors: Hervé F , Martin M T , Rajkowski K , Dessen P , Cittanova N
Published: August 2015

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.
The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles
Authors: Dawson A P , Hills G , Comerford J G
Published: August 2015

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), if added before GTP, blocks both Ca2+ efflux promoted by GTP and the effect of GTP on enhancement of inositol 1,4,5-triphosphate (IP3)-promoted Ca2+ release from preloaded microsomal vesicles. If, however, GTP[S] is added after GTP, it does not reverse the Ca2+ efflux promoted by GTP, nor does it inhibit IP3-promoted Ca2+ release. 2. The effect of GTP in enhancing IP3-promoted Ca2+ release is maintained after washing the microsomal vesicles free of added GTP. After this treatment, enhancement of IP3-promoted Ca2+ efflux can be observed in the absence of poly(ethylene glycol). 3. Electron microscopy shows that during GTP treatment of microsomal vesicles there is rapid production of very large vesicular structures, apparently produced by fusion of smaller vesicles. 4. Light-scattering changes are detectable during the fusion process. 5. Both Ca2+ efflux promoted by GTP and the enhancement of IP3-promoted Ca2+ release seen in the presence of GTP can probably be attributed to GTP-dependent vesicle fusion.
Prostaglandin E1 and forskolin antagonize C-kinase activation in the human platelet
Authors: de Chaffoy de Courcelles D , Roevens P , Van Belle H
Published: August 2015

In human platelets, prostaglandin E1 and forskolin inhibit 5-hydroxytryptamine-induced phospholipase C, C-kinase and myosin light-chain kinase activity in a concentration-dependent way. Phospholipase C activation, however, was only partly inhibited, and this at higher concentrations than the protein kinases. Direct activation of the C kinase either by exogenous synthetic diacylglycerol or by 12-O-tetradecanoylphorbol 13-acetate was antagonized by prostaglandin E1 and forskolin. Since C-kinase activation is one of the key events in excitatory signal transduction in the platelets, we suggest that the inhibitory effect of agents that increase platelet cyclic AMP on platelet secretion and aggregation might reside in their capacity to antagonize C-kinase activity.
Fructose 2,6-bisphosphate in rat erythrocytes. Inhibition of fructose 2,6-bisphosphate synthesis and measurement by glycerate 2,3-bisphosphate
Authors: Sobrino F , Rider M H , Gualberto A , Hue L
Published: August 2015

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.
Induction of high-density-lipoprotein receptors in rat corpus luteum by human choriogonadotropin. Evidence of protein synthesis de novo
Authors: Ghosh D K , Menon K M
Published: August 2015

The present studies investigated the specific binding of 125I-labelled high-density lipoprotein (125I-HDL) to plasma membranes. Golgi, rough endoplasmic reticulum and mitochondria/lysosomes, prepared from ovaries of rats injected with human choriogonadotropin (hCG) or 0.9% NaCl. Treatment in vivo with hCG resulted in 2-3-fold induction of 125I-HDL binding activity in all the subcellular organelles. The specific binding of HDL to various subcellular organelles was dependent on the amount of protein, lipoprotein concentration and incubation time. Equilibrium-binding studies revealed comparable Kd values (13-22 micrograms of HDL protein/ml) for HDL binding in all the subcellular organelles tested. Treatment with cycloheximide (2.0 mg/kg body wt.) before hCG administration abolished the induction of HDL receptors, suggesting the involvement of a protein-synthesis-dependent process in receptor induction. Analysis of equilibrium dissociation constants (Kd) for 125I-HDL binding in membranes from hCG-, cycloheximide-and saline-treated animals suggests that the increase in binding was due to an increase in the number of binding sites rather than a change in the affinity. Additionally, pretreatment with tunicamycin, an inhibitor of N-linked glycosylation, had no effect on hCG-mediated receptor induction, suggesting that glycosylation of the receptor may not be necessary for the interaction of HDL with its receptors.
Nitrogenase of Klebsiella pneumoniae. Rhodanese-catalysed restoration of activity of the inactive 2Fe species of the Fe protein
Authors: Pagani S , Eldridge M , Eady R R
Published: August 2015

The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.
The effect of sn-2 fatty acid substitution on phospholipase C enzyme activities
Authors: Bomalaski J S , Clark M A
Published: August 2015

The human monocyte cell line U937 expresses phospholipase A2 and phospholipase C activities and produces eicosanoids. The phospholipase C (PLC) activity exhibits substrate preference for phosphatidyl-choline (PC), rather than phosphatidylinositol or phosphatidylethanolamine. In order to characterize the PLC activity found in these cells, the effects of substitution of the sn-2 fatty acid on this activity were examined. PC substrates with palmitic acid (PC-2P), oleic acid (PC-2O), arachidonic acid (PC-2A) and linoleic acid (PC-2L) at the sn-2 position were used. The sn-1 fatty acid was palmitic acid. PC-2L and PC-2A with the longer-chain less-saturated fatty acids linoleic acid and arachidonic acid esterified at sn-2 were found to be better substrates for PLC activity than PC-2P or PC-2O in these cells. This preference was maintained even when substrate phospholipid was solubilized in non-ionic, anionic, cationic and zwitterionic amphiphiles. Furthermore, when a 500-fold excess of 1,2-diolein or 1,2-dipalmitin was added to the reaction, the specificity of the PLC activity for PC-2A and PC-2L remained unchanged. When similar experiments were performed with phosphatidylinositol as a substrate, we did not observe any effect when the sn-2 position was altered. These data show that the fatty acid constituent at the sn-2 position affects the observed PLC activity when phosphatidylcholine, but not phosphatidylinositol, is used as a substrate by these cells.
An NAD+-dependent alanine dehydrogenase from a methylotrophic bacterium
Authors: Bellion E , Tan F
Published: August 2015

A study was made of the NAD+-dependent alanine dehydrogenase (EC 1.4.1.1) elaborated by the methylotrophic bacterium Pseudomonas sp. strain MA when growing on succinate and NH4Cl. This enzyme was purified 400-fold and was found to be highly specific for NH3 and NAD+; however, hydroxypyruvate and bromopyruvate, but not alpha-oxoglutarate or glyoxylate, could replace pyruvate to a limited extent. The Mr of the native enzyme was shown to be 217,000, and electrophoresis in SDS/polyacrylamide gels revealed a minimum Mr of 53,000, suggesting a four-subunit structure. The enzyme, which has a pH optimum of 9.0, operated almost exclusively in the aminating direction in vitro. It was induced by NH3 or by alanine, and was repressed by growth on methylamine or glutamate. It is suggested that this enzyme has two roles in this organism, namely in NH3 assimilation and in alanine catabolism.
Biochemical characterization of individual normal, floppy and rheumatic human mitral valves
Authors: Lis Y , Burleigh M C , Parker D J , Child A H , Hogg J , Davies M J
Published: August 2015

Human mitral valves (32 floppy and 17 rheumatic) obtained at surgery were analysed and compared with 35 normal (autopsy) valves. Total amounts of collagen, proteoglycan and elastin were increased approx. 3-fold in floppy and rheumatic valves. The water content of rheumatic cusps was lower than normal. The most significant changes in floppy valves were the 59% increase in mean value of the proteoglycan content, a large increase in the ease of extractability of proteoglycans from 26.7 to 57.2% of the total and a 62% increase in mean value of the elastin content in the anterior cusps. Normal human mitral valve cusps contained a mean proportion of 29.3 (and chordae 26.6) type III collagen (as % of total types III + I collagen), the values increasing significantly to 33.2 and 36.3% respectively in chronic rheumatic disease. The ratio observed in floppy valves depended on the extent of secondary surface fibrosis, which could be demonstrated histologically; in valve cusps with considerable secondary fibrosis, the percentage of type III increased significantly (to 34.4%), whereas it decreased significantly (to 25.2%) when fibrosis was negligible. It is concluded that the ratio of collagen types in floppy valves reflects the extent of secondary fibrosis rather than the pathogenesis of the disrupted collagen in the central core of the valve.
Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts
Authors: Boegheim J P J , Dubbelman T M A R , Mullenders L H F , Van Steveninck J
Published: August 2015

Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On ‘post-incubation’, the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.
Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors
Authors: Mauco G , Dajeans P , Chap H , Douste-Blazy L
Published: August 2015

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.
Properties of highly viscous gels formed by neurofilaments in vitro. A possible consequence of a specific inter-filament cross-bridging
Authors: Leterrier J F , Eyer J
Published: August 2015

Neurofilaments freshly isolated from bovine spinal cord form a reversible gel in vitro, consisting of nearly parallel and interlinked filaments organized in bundles. This phenomenon is obtained above a critical neurofilament concentration and is highly sensitive to denaturation. No gelation occurs with neurofilaments reconstituted from urea-solubilized subunits. The velocity of the gelation kinetics, optimum at a slightly acidic pH, is inhibited by low and high ionic strength and activated by millimolar concentrations of Mg2+ and other bivalent cations. No protein other than the purified neurofilament preparation itself (80-95% neurofilament triplet) is necessary for the formation of a gel. However, purified cytoskeletal proteins from microtubules and neurofilaments influence the viscosity of the native preparation. These observations suggest a reticulation in vitro between neurofilaments, dependent upon a fragile conformation of the polymers and possibly mediated through the high-Mr neurofilament subunits (200 kDa and 150 kDa). The significance of these results is discussed with regard to the inter-neurofilament cross-bridging in situ involving the 200 kDa subunit described by Hirokawa, Glicksman & Willard [(1984) J. Cell Biol. 98, 1523-1536].
Structure of two new aminophospholipids from Methanobacterium thermoautotrophicum
Authors: Kramer J K G , Sauer F D , Blackwell B A
Published: August 2015

The methanogenic bacterium Methanobacterium thermoautotrophicum (A.T.C.C. 29183) was shown to contain two new aminophospholipids. These are 2-aminoethyl phosphate ester of diphytanylglycerol diether and a sugar containing bisdiphytanyldiglycerol tetraether. The two aminophospholipids were stable to acid methanolysis except for the sugar on the bisdiphytanyldiglycerol tetraether. Strong acid (6 M-HCl) hydrolysed the alkyl ether and aminophosphate ester bonds. The structure of the phosphate linkage was demonstrated by 31P n.m.r., and the 2-ethanolamine structure was elucidated by 1H- and 13C-n.m.r. spectroscopy and by fast-atom-bombardment m.s.
The effect of concanavalin A on the rat electro-olfactogram at various odorant concentrations
Authors: Shirley S G , Polak E H , Edwards D A , Wood M A , Dodd G H
Published: August 2015

We have studied the effect of concanavalin A (Con A) on the rat electro-olfactogram response to several odorants. Each odorant was applied over a range of concentrations. For hydrophobic odorants whose response was affected by Con A, the diminution in response was maximal at odorant concentrations of about 1 microM in the olfactory mucus. The (odour) concentration-dependence of the change is compatible with the idea that Con A inactivates one or more types of olfactory receptor that normally bind odorants with dissociation constants of the order of 100 nM. With hydrophilic odorants we had to apply concentrations very much higher than this to elicit any response from the system. At these high concentrations we could observe Con A-induced diminutions in response.
Age-specific nuclear proteins in the nematode worm Caenorhabditis elegans
Authors: Meheus L A , Van Beeumen J J , Coomans A V , Vanfleteren J R
Published: August 2015

The nematode worm Caenorhabditis elegans is known to undergo characteristic morphological as well as physiological signs of senescence. Two-dimensional gel electrophoresis shows that alterations also occur in the pattern of the nuclear proteins as a function of age. Non-histone proteins whose level exhibits a steep fall with age are egg-specific and not involved in senescence. However, a distinct set of non-histones accumulates with age and can be considered as senescence markers. Some of these are glycoproteins, as shown by their concanavalin A-binding properties. One age-specific polypeptide, called ‘protein S-28’, was further characterized by peptide mapping and determination of its N-terminal amino acid sequence.
Metabolism of inositol 1,4,5-trisphosphate in permeabilized rat aortic smooth-muscle cells. Dependence on calcium concentration
Authors: Rossier M F , Capponi A M , Vallotton M B
Published: August 2015

The metabolism of [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) was studied in permeabilized rat aortic smooth-muscle cells. Addition of [3H]Ins(1,4,5)P3 to the leaky cells led to formation of several labelled metabolites. Amounts of [3H]inositol bisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate ([3H]InsP4) reached a maximum within 2 min of incubation, whereas production of [3H]inositol monophosphate and [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3) was delayed. Formation of InsP4 and Ins(1,3,4)P3 was Ca2+-sensitive in the physiological intracellular range (0.06-5 microM), showing a maximum at 1 microM-Ca2+. A correlation between the formation of InsP4 and that of Ins(1,3,4)P3 was observed, suggesting that the former is the precursor of the latter. These results suggest that, in vascular smooth-muscle cells, Ins(1,4,5)P3 is metabolized via two distinct pathways: (1) a dephosphorylation pathway, leading to formation of inositol bis- and mono-phosphate; and (2) a Ca2+-sensitive phosphorylation/dephosphorylation pathway, involving formation of InsP4 and leading to formation of Ins(1,3,4)P3.
Metal constitution of metallothionein influences inhibition of δ-aminolaevulinic acid dehydratase (porphobilinogen synthase) by lead
Authors: Goering P L , Fowler B A
Published: August 2015

This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd, Zn-thionein, although minimal binding of 203Pb to kidney Cd, Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd, Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd, Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd, Zn-thionein much greater than kidney Cd, Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd, Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.
Sex-specific constitutive expression of the pre-neoplastic marker glutathione S-transferase, YfYf, in mouse liver
Authors: McLellan L I , Hayes J D
Published: August 2015

Hepatic glutathione S-transferase isoenzyme content has been investigated in both sexes of three inbred strains of mice (DBA/2, C3H/He, C57BL6). A polypeptide (Mr 24,800), which is immunologically related to Yf purified from rat lung, was found to be expressed as a major form in all male mouse livers but represented only a minor enzyme form in female mouse liver. Glutathione S-transferases comprising subunits with molecular masses of 25,800 (Ya) or 26,400 (Yb) were present in males and females of the three strains under investigation. Cytosolic isoenzymes from all strains and sexes were purified to apparent homogeneity and no significant inter-strain differences in the properties of the individual forms were observed. In addition, no differences were detected in the microsomal glutathione S-transferase content of the different strains or sexes.
Characterization of structural component of cell walls of alkalophilic strain of Bacillus sp. C-125. Preparation of poly(γ-l-glutamate) from cell wall component
Authors: Aono R
Published: August 2015

The cell wall of an alkalophilic strain of Bacillus sp. C-125 is composed of A1 gamma-peptidoglycan, a teichuronic acid and an unknown acidic polymer composed of glutamic acid and glucuronic acid, of which the molar ratio is approx. 4-5:1. Poly(gamma-L-glutamate) was prepared from the acidic polymer by removal of almost all of the glucuronic residues with trifluoromethanesulphonic acid treatment and purified chromatographically. The Mr of the polyglutamate preparation was estimated to be 14,000 by gel chromatography, or 43,000 on the basis of the content of N-terminal acid residues. The acidic polymer found in the cell wall of the organism was concluded to be a polyglutamate substituted with (oligo)glucuronic acid residues or a complex composed of two kinds of polymers (polyglutamate and polyglucuronate).
Characterization of Ca2+ uptake and release by vesicles of skeletal-muscle sarcoplasmic reticulum
Authors: McWhirter J M , Gould G W , East J M , Lee A G
Published: August 2015

Homogenization of muscle gives a preparation of sealed vesicles derived from the sarcoplasmic reticulum. In the presence of ATP these vesicles will initially accumulate Ca2+ from the external medium and then spontaneously release this Ca2+ in two phases, an initial slow phase and a faster second phase. By measuring ATP concentrations in parallel with measurements of external Ca2+ concentrations we have shown that the second phase of release occurs when the added ATP has been exhausted, but that the first phase of release occurs in the presence of ATP. A similar pattern of uptake and release has been observed in the presence of acetyl phosphate, showing that ADP generated by ATP hydrolysis is not essential for the release process. The temperature-dependence of both phases of release is similar to the temperature-dependence of ATPase activity. Release is dependent on pH over the same pH range as affects binding of Ca2+ to the ATPase. Therefore we propose that Ca2+ release from vesicles of sarcoplasmic reticulum actively loaded with Ca2+ is mediated by the same Ca2+ + mg2+-activated ATPase as is responsible for uptake of Ca2+.
Studies on chemical modification of cold agglutinin from the snail Achatina fulica
Authors: Sarkar M , Mitra D , Sen A K
Published: August 2015

The cold agglutinin isolated from the albumin gland of the snail Achatina fulica was modified with various chemical reagents in order to detect the amino acids and/or carbohydrate residues present in its carbohydrate-binding sites. Treatment with reagents considered specific for modification of lysine, arginine and tryptophan residues of the cold agglutinin did not affect the carbohydrate-binding activity of the agglutinin. Modification of tyrosine residues showed some change. However, modification with carbodiimide followed by α-aminobutyric acid methyl ester causes almost complete loss of its binding activity, indicating the involvement of aspartic acid and glutamic acid in its carbohydrate-binding activity. The carbohydrate residues of the cold agglutinin were removed by beta-elimination reaction, indicating that the sugars are O-glycosidically linked to protein part of the molecule. Removal of galactose residues from the cold agglutinin by the action of beta-galactosidase indicated that the galactose molecules are beta-linked. These carbohydrate-modified glycoproteins showed a marked change in agglutination property, i.e. they agglutinated rabbit erythrocytes at both 10 degrees C and 25 degrees C, indicating that the galactose residues of the glycoprotein play an important role in the cold-agglutination property of the glycoprotein. The c.d. data showed the presence of an almost identical type of random-coil conformation in the native cold agglutinin at 10 degrees C and in the carbohydrate-modified glycoprotein at 10 degrees C and 25 degrees C. This particular random-coil conformation is essential for carbohydrate-binding property of the agglutinin.
Mechanisms of leucine- and theophylline-stimulated insulin biosynthesis in isolated rat pancreatic islets
Authors: Welsh N , Welsh M , Steiner D F , Hellerström C
Published: August 2015

To extend previous observations on the mechanisms of translational regulation of insulin biosynthesis [Welsh, Scherberg, Gilmore & Steiner (1986) Biochem. J. 235, 459-467], we have now compared the intracellular distributions of insulin mRNA after stimulation of insulin biosynthesis by glucose, leucine or theophylline. In comparison with low glucose (3.3 mM) only, the presence of 10 mM-leucine + 3.3 mM-glucose resulted in the transfer of insulin mRNA from the pool of the uninitiated mRNA to the free polysome/monosome fraction and an increase in the amount of insulin mRNA associated with the microsomal fraction. Islets exposed to 5 mM-theophylline + 3.3 mM-glucose also showed a decreased content of uninitiated insulin mRNA in the cytosol, but these islets showed no increase in insulin mRNA in the microsomal fraction. These results suggest that leucine, a nutrient stimulant of insulin biosynthesis, acts essentially by the same mechanisms as those of glucose, whereas theophylline acts only to stimulate initiation rates.
Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri)
Authors: Babin P J
Published: August 2015

I have previously described [Babin (1987) J. Biol. Chem. 262, 4290-4296] the apolipoprotein composition of the major classes of trout plasma lipoproteins. The present work describes the use of an isopycnic density gradient centrifugation procedure and sequential flotation ultracentrifugation to show: (1) the presence of intermediate density lipoproteins (IDL) in the plasma, between 1.015 and 1.040 g/ml; (2) the existence of a single type of Mr 240,000 apoB-like in the low density lipoproteins (LDL, 1.040 less than p less than 1.085 g/ml); (3) the presence of apoA-I-like (Mr 25,000) in the densest LDL; (4) the adequacy of 1.085 g/ml as a cutoff between the LDL and high density lipoproteins (HDL); (5) the accumulation of Mr 55,000 and 76,000 apolipoproteins and apoA-like apolipoproteins in the 1.21 g/ml infranatant. The fractionation of trout lipoprotein spectrum thus furnishes the distribution of the different lipoprotein classes and leads to the description of the constituent apolipoproteins, which account for about 36% of circulating plasma proteins in this species.
Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase does not respond to ubiquinone uptake in cultured cells
Authors: Maltese W A , Aprille J R , Green R A
Published: August 2015

The cellular content of ubiquinone was increased approx. 10-fold by incubation of neuroblastoma cells in medium containing exogenous ubiquinone. Under these conditions the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, assayed after preincubation of cell homogenates with or without fluoride, was not suppressed. Similar results were obtained with human skin fibroblast cultures to which free ubiquinone or low-density lipoprotein-ubiquinone complex had been added. Consistent with the lack of suppression of HMG-CoA reductase, the rate of incorporation of [1-14C] acetate into ubiquinone was not diminished in cells exposed to exogenous ubiquinone. Measurements of [3H]mevalonolactone incorporation into cellular ubiquinones indicated that exogenous ubiquinone did not affect ubiquinone synthesis at a point in the pathway distal to the formation of mevalonate. The results suggest that cultured mammalian cells lack an end-product ‘feedback’ mechanism for regulation of HMG-CoA reductase in response to ubiquinone uptake.