bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on myelin-basic-protein methylation during mouse brain development
Authors: Chanderkar L P , Paik W K , Kim S
Published: August 2015

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of ‘acid extracts’ and ‘breakthrough’ fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.
Sequence analysis and transformation by the catabolic 3-dehydroquinase (QUTE) gene from Aspergillus nidulans
Authors: Da Silva A J , Whittington H , Clements J , Roberts C , Hawkins A R
Published: August 2015

The induction of catabolic 3-dehydroquinase by quinic acid in Aspergillus nidulans has been shown to involve transcriptional control and yields a single major 0.8 kb mRNA. The nucleotide sequence of the catabolic 3-dehydroquinase QUTE gene has been determined and contains a single uninterrupted open reading frame of 462 bases encoding a 16,505 Da protein of 153 residues. Comparison with the corresponding QA2 gene of Neurospora crassa reveals the absence of 75 nucleotides encoding 25 amino acids from the centre of the QUTE gene of A. nidulans and the presence of 21 additional nucleotides at its 3′ end. There is no nucleotide or amino acid homology between these two elements. A 16 bp inverted repeat (5′ GGCAGAGCGTTCTGCC) shows similarity to such repeats found in other fungal promoters. The functional integrity of the QUTE gene was demonstrated by the transformation of a qutE mutant strain which regains growth on quinic acid as sole carbon source. Four of the twelve transformed strains examined contained vector sequences integrated at the qutE locus, and these strains all exhibited normal regulation of 3-dehydroquinase even when 16 copies of the QUTE gene were present.
Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release
Authors: Shoshan-Barmatz V
Published: August 2015

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5′-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).
NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5′-deiodinase. Demonstration by alkylation with iodoacetamide
Authors: Das A K , Hummel B C W , Walfish P G
Published: August 2015

We have assessed a previously proposed mechanism mediating 5′-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5′-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5′-deiodinase correlated with the amount of available (unalkylated) Fraction B.
The structures and proteolytic specificities of autolysed human thrombin
Authors: Chang J Y
Published: August 2015

Three Arg/Lys-Xaa bonds in the B-chain of human alpha-thrombin were found to be the major autolytic sites. Under the conditions of 1 mg of alpha-thrombin/ml in 50 mM-ammonium bicarbonate solution at 25 degrees C, the 50% cleavage times of Lys-Gly (residues 154-155), Arg-Tyr (residues 70-71) and Arg-Asn (residues 73-74) were 32 h, 72 h and 96 h respectively. Fragments generated from these three major autolytic sites were purified and analysed. In addition, minor and random autolytic cleavages occurred simultaneously that eventually led to the complete breakdown of the enzyme. These results reveal several novel aspects about the process of autolysis and the structure of autolysed human thrombin. It identifies a major autolytic site at Arg-Tyr (residues 70-71) that has not been previously reported. It demonstrates that beta-thrombin is not an obligatory intermediate during the process of conversion of alpha-thrombin into gamma-thrombin. There exists a new form of autolysed thrombin, designated as beta'-thrombin (with cleavage at Lys-Gly only), which also serves as the intermediate in the conversion of alpha-thrombin into gamma-thrombin. It shows that autolysis of human alpha-thrombin does not proceed in an absolutely clear-cut manner. Numerous minor cleavages, which amount to approx. 20% of the three major autolytic sites, occur simultaneously. It is the first time that several autolytic sites of human alpha-thrombin have been quantitatively analysed, and that it has been shown that formation of beta-, beta'- and gamma-thrombins can be quantitatively followed by the h.p.l.c. method. Furthermore, the data demonstrate that alpha-thrombin and the autolysed thrombin (mixture of beta-, beta'- and gamma-thrombins) have comparable proteolytic activity and specificity towards various sizes of non-fibrinogen polypeptide substrates with relative molecular masses ranging from 3000 to 25,000.
Light stimulates the rapid formation of inositol trisphosphate in squid retinas
Authors: Szuts E Z , Wood S F , Reid M S , Fein A
Published: August 2015

To test the hypothesis that inositol trisphosphate (InsP3) mediates adaptation and excitation in invertebrate photoreceptors, we measured its formation on a rapid time scale in squid retinas. For squid, excitation and adaption occurs within 0.1 and 1-2 s respectively. We could detect an elevation in InsP3 within 200 ms of a bright flash. This increase is about 240% over dark basal levels and is maintained for at least 2 min after a flash. The increase probably occurs in the photoreceptors, which are the only neurons in squid retinas. Analysis by h.p.l.c. indicates that the light-regulated isomer is Ins(1,4,5)P3, which is formed by the hydrolysis of phosphatidylinositol bisphosphate (PtdInsP2).
A novel acyl-CoA-binding protein from bovine liver. Effect on fatty acid synthesis
Authors: Mogensen I B , Schulenberg H , Hansen H O , Spener F , Knudsen J
Published: August 2015

Bovine liver was shown to contain a hitherto undescribed medium-chain acyl-CoA-binding protein. The protein co-purifies with fatty-acid-binding proteins, but was, unlike these proteins, unable to bind fatty acids. The protein induced synthesis of medium-chain acyl-CoA esters on incubation with goat mammary-gland fatty acid synthetase. The possible function of the protein is discussed.
Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry
Authors: Nissim I , Yudkoff M , Segal S
Published: August 2015

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.
Two globin strains in the giant annelid extracellular haemoglobins
Authors: Gotoh T , Shishikura F , Snow J W , Ereifej K I , Vinogradov S N , Walz D A
Published: August 2015

The constituent polypeptide chains I, II, III and IV of the giant extracellular haemoglobin of the oligochaete Lumbricus terrestris were isolated by mono Q ion-exchange chromatography and C8 reverse-phase chromatography. The N-terminal amino acid sequences of Lumbricus chains I, III and IV were determined and aligned with those of Lumbricus chain II and the four chains of the extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus. Three invariant amino acid residues, Cys-7, Val-15 and Trp-19, were found to occur in the N-terminal segments (17-22 residues) of the eight chains of Lumbricus and Tylorrhynchus haemoglobins. In addition, it was found that the eight sequences could be separated into two groups: ‘A’, consisting of Lumbricus chains I and II and Tylorrhynchus chains I and IIA, having invariant Lys-14 and Lys-16, and ‘B’, consisting of Lumbricus chains III and IV and Tylorrhynchus IIB and IIC, having invariant Cys-6, Ser-8 and Asp-11. This result suggests that there are two strains of globin chain in the annelid extracellular haemoglobins.
Protein phosphorylation in rat mammary acini and in cytosol preparations in vitro Phosphorylation of acetyl-CoA carboxylase is unaffected by cyclic AMP
Authors: Clegg R A , West D W , Aitchison R E D
Published: August 2015

Phosphorylation of soluble proteins in rat mammary acinar cells was investigated. When phosphorylation proceeded in intact cells, in the presence of [32P]Pi, the major non-casein phosphoproteins, including acetyl-CoA carboxylase, were unresponsive to incubation conditions that caused major increases in the intracellular concentration of cyclic AMP. The overall 32P specific radioactivity (c.p.m./microgram of protein) of acetyl-CoA carboxylase, assessed after affinity purification of the enzyme with avidin-Sepharose, was unchanged by incubation under such conditions. Furthermore, the distribution of 32P among tryptic phosphopeptides of the enzyme, resolved by reversed-phase h.p.l.c., was not altered by cyclic AMP-increasing treatments of the acinar cells. When cytosol fractions were incubated with [gamma-32P]ATP, some phosphoproteins responded to the addition of micromolar concentrations of dibutyryl cyclic AMP or cyclic AMP by undergoing an enhancement of phosphate incorporation. In these experiments in vitro, protein phosphatase activity did not make a major contribution to the net phosphorylation of individual phosphoproteins, and acetyl-CoA carboxylase was not prominent among the phosphoproteins identified after short (less than 1 min) incubations of cytosols with [gamma-32P]ATP. The resistance of protein phosphorylation to variations in the cyclic AMP concentration in intact mammary epithelial cells, demonstrated by this work, is one of several mechanisms that ensure the pleiotropic refractoriness of those cells to agents which normally cause a stimulation of adenylate cyclase activity in hormone-sensitive cells.
Altered interactions between lipogenesis and fatty acid oxidation in regenerating rat liver
Authors: Schofield P S , Sugden M C , Corstorphine C G , Zammit V A
Published: August 2015

The concentrations of malonyl-CoA, citrate, ketone bodies and long-chain acylcarnitine were measured in freeze-clamped liver samples from fed or starved normal, partially hepatectomized or sham-operated rats. These parameters were used in conjunction with measurements of the concentration of plasma non-esterified fatty acids and the rates of hepatic lipogenesis to obtain correlations between rates of fatty acid delivery to the liver, lipogenesis and fatty acid oxidation to ketone bodies and CO2. These correlations indicated that the development of fatty liver after partial hepatectomy is due to an increased partitioning of long-chain acyl-CoA towards acylglycerol synthesis and away from acylcarnitine formation. However, this did not appear to be due to an altered relationship between hepatic malonyl-CoA concentration and acylcarnitine formation. For any concentration of long-chain acylcarnitine, the concentrations of both hepatic and blood ketone bodies were significantly lower in partially hepatectomized rats than in normal or sham-operated animals. This indicated that a lower proportion of the product of beta-oxidation was used for ketone-body formation and more for citrate synthesis in the regenerating liver, especially during the first 24 h after resection. This inference was supported by the changes in hepatic citrate concentrations observed. The high rates of lipogenesis that occurred in the liver remnant were accompanied by an altered relationship between lipogenic rate and hepatic malonyl-CoA concentration, such that much lower concentrations of malonyl-CoA were associated with any given rate of lipogenesis. These adaptations are discussed in relation to the requirements by the remnant for high rates of energy formation through the tricarboxylic acid cycle during the first 24 h after resection, and the possibility that cycling between fatty acid oxidation and synthesis may occur to a greater degree in regenerating liver.
Ketone-body metabolism after surgical stress or partial hepatectomy. Evidence for decreased ketogenesis and a site of control distal to carnitine palmitoyltransferase I
Authors: Schofield P S , French T J , Sugden M C
Published: August 2015

Rats were subjected to laparotomy, or laparotomy and partial hepatectomy, at 0-48 h before administration of water or medium-chain-length triacylglycerol, having been starved post-operatively. Functional hepatectomies were performed at intervals after the intragastric load. Blood ketone-body concentrations after medium-chain triacylglycerol administration and/or functional hepatectomy of these rats were compared with values obtained in starved control rats. Decreased ketonaemia in response to medium-chain triacylglycerol was observed for up to 48 h after partial hepatectomy and at 1 and 2 h after laparotomy, but not at 24 or 48 h after laparotomy. Rates of ketone-body clearance after functional hepatectomy were unaffected by prior laparotomy or partial hepatectomy. Ketonaemia after medium-chain-triacylglycerol administration was only partially blocked by inhibition of CPT I (carnitine palmitoyltransferase I). The results demonstrate sustained effects of partial hepatectomy and short-term effects of surgical stress to decrease ketonaemia via inhibition of ketogenesis at site(s) distal to CPT I.
Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of α-amanitin-resistant and -sensitive enzymes
Authors: Earnshaw D L , Beebee T J C , Gutteridge W E
Published: August 2015

We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.
The rapid alteration by tri-iodo-l-thyronine in vivo of both the ADP/O ratio and the apparent H+/O ratio in hypothyroid-rat liver mitochondria
Authors: Crespo-Armas A , Mowbray J
Published: August 2015

Mitochondria from the livers of thyroidectomized rats have a lowered ADP/O ratio, which can be restored to normal within 15 min after intravenous injection of a near-physiological dose of tri-iodothyronine. Thyroidectomy lowered the measured delta pH, which appears to be compensated by a rise (not statistically significant) in the membrane potential, so that the protonmotive force is unaltered. A simple simulation technique is described for use in estimating H+/O ratios by the oxygen-pulse technique, which circumvents the problem that this ratio can be seriously underestimated because of re-uptake of protons from the bulk phase by the mitochondria before their expulsion is complete. By this procedure the H+/O ratio of hypothyroid mitochondria is shown to be lowered by the same factor as the ADP/O ratio, and both these ratios are very rapidly restored in parallel by hormone administration. Although these findings could be consistent with a proposal that tri-iodothyronine rapidly modulates by some mechanism the efficiency of the respiratory-chain-linked proton pumps, the kinetic properties of the proton exchange suggest that the bulk-phase protons measured may not reflect faithfully those that drive the ATP synthetase.
The insulin- and glucagon-stimulated ‘dense-vesicle’ high-affinity cyclic AMP phosphodiesterase from rat liver. Purification, characterization and inhibitor sensitivity
Authors: Pyne N J , Cooper M E , Houslay M D
Published: August 2015

The hormone-stimulated ‘dense-vesicle’ cyclic AMP phosphodiesterase was solubilized as a proteolytically ‘clipped’ species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native ‘dense-vesicle’ enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified ‘clipped’ enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].
The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages
Authors: Hancock J T , Jones O T
Published: August 2015

Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.
Enzymic dephosphorylation of d-myo-inositol 1,4-bisphosphate in rat brain
Authors: Delvaux A , Erneux C , Moreau C , Dumont J E
Published: August 2015

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.
The regulation of lipogenesis in vivo in the lactating mammary gland of the rat during the starved-refed transition. Studies wtih acarbose, a glucosidase inhibitor
Authors: Mercer S W , Williamson D H
Published: August 2015

Depression of carbohydrate digestion by oral administration of acarbose, a glucosidase inhibitor, led to a 75% inhibition of the re-activation of lipogenesis in vivo in the mammary gland of 18 h-starved lactating rats refed with 5 g of chow diet. Rates of [1-14C]glucose incorporation in vitro into lipid and CO2 in mammary-gland acini isolated from refed animals were elevated compared with acini from starved rats, but acarbose treatment completely prevented this stimulation. Gastric intubation of glucose led to a large stimulation of lipogenesis in the mammary gland of starved lactating rats, similar to that induced by refeeding with chow diet; this was dependent on the amount of glucose given and the time elapsed between glucose administration and injection of 3H2O for the measurement of lipogenesis. The switch-on of lipogenesis in the mammary gland of starved lactating rats, by refeeding or by intubation of glucose, was associated with a decrease in the ratio of [glucose 6-phosphate]/[fructose 1,6-bisphosphate] in the gland, indicative of an increase in phosphofructokinase activity. A time-course study revealed that the ratio decreased rapidly over the first 30 min of chow refeeding, after which a large surge in lipogenesis was seen. Acarbose, given 25 min after the onset of refeeding, led to a stepwise increase in the ratio, in parallel with the observed decrease in lipogenic activity. It is concluded that the control of lipogenesis in the mammary gland is closely linked to the availability of dietary carbohydrate. An important site of regulation of lipogenesis in the gland appears to be at the level of phosphofructokinase. A possible role of insulin in the regulation of phosphofructokinase activity, and the acute modulation of insulin-sensitivity in the gland during the starved-refed transition, are discussed.
Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein
Authors: Baltz M L , Rowe I F , Caspi D , Turnell W G , Pepys M B
Published: August 2015

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.
Zymosan-induced release of inositol phosphates at resting cytosolic Ca2+ concentrations in macrophages
Authors: Moscat J , Herrero C , Garcia-Barreno P , Municio A M
Published: August 2015

Hydrolysis of polyphosphoinositides by phosphodiesterase has been demonstrated to be involved in the control of cytosolic Ca2+ concentrations. The stimulation of Ca2+ ionophores of the release of inositol phosphates in macrophages, and other cells, together with the Ca2+ requirements for zymosan-induced phospholipase C activation, make unclear the relationship between Ca2+ mobilization and polyphosphoinositide hydrolysis. The results in the present paper strongly suggest that, for zymosan-induced phospholipase C activation, a previous increase in cytosolic Ca2+ is not a required event. These results also show that zymosan-activated release of inositol phosphates may be mediated by a guanine-nucleotide-binding protein.
Inhibition of intracellular protein degradation by ethanol in perfused rat liver
Authors: Pösö A R , Surmacz C A , Mortimore G E
Published: August 2015

Ethanol (50 mM) inhibited proteolysis in the perfused rat liver during stringent amino acid deprivation and also in the presence of normal and 10 times normal concentrations of plasma amino acids. The concentration-response curve of ethanol reached a plateau after 5 mM in both the presence and the absence of normal plasma amino acids, suggesting inhibition by oxidation products of ethanol. Intracellular glutamine, tyrosine and proline increased in concentration with ethanol, but the increases were too small to explain the observed inhibition of proteolysis. The uptake of 125I-asialofetuin was slightly decreased and the output of ammonia increased in the presence of ethanol. These, together with a significant suppression of basal proteolysis in the presence of amino acids, suggest that lysosomal function was directly affected. Electron-microscopic examination of lysosomal components showed that the aggregate volume of autophagosomes (initial vacuoles) were significantly smaller in livers perfused with ethanol than in controls. However, the equivalent volume of autolysosomes (degradative vacuoles) was the same in both groups. According to these results, ethanol inhibits protein degradation in the liver by two discrete mechanisms: one decreasing the formation of autophagic vacuoles and the other involving lysosomotropic inhibition, possibly via ammonia.
Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA
Authors: Jackson M R , McCarthy L R , Harding D , Wilson S , Coughtrie M W H , Burchell B
Published: August 2015

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).
Effects of stimulation of muscarinic and of β-catecholamine receptors on the intracellular distribution of protein kinase C in guinea pig exocrine glands
Authors: Machado-de Domenech E , Söling H D
Published: August 2015

Stimulation of exocrine cells via muscarinic receptors is associated with an activation of protein kinase C [Padel & Söling (1985) Eur. J. Biochem. 151, 1-10]. We show here that stimulation of isolated parotid gland lobules with 8 X 10(-6) M-carbamoylcholine leads to a translocation of protein kinase C from the cytosolic to the particulate compartment within 30 s (25% and 45% of total activity recovered in the particulate fraction of controls and stimulated samples respectively). The specific enzyme activity in the particulate fraction increased to 169% of the corresponding control value. After 10 min the changes started to reverse and, after 30 min, cytosolic protein kinase C was higher in stimulated than in unstimulated lobules. Isoproterenol (2 X 10(-5) M) stimulated the release of amylase more than did carbamoylcholine, but did not significantly affect intracellular distribution of protein kinase C during the observation time of 30 min. In isolated pancreatic lobules a significant carbamoylcholine-mediated translocation of protein kinase C into the particulate fraction could be observed after 5 and 20 min, but not after 1 min. After 5 min the specific enzyme activity in the particulate fraction had increased to 153% of the corresponding controls. The corresponding decrease (-38%) in the specific enzymic activity of cytosolic protein kinase C stayed constant up to 30 min. In isolated parotid gland lobules alpha-amylase secretion proceeded at a linear rate already during the first 1 min of stimulation, whereas in pancreatic lobules a measurable rate of alpha-amylase secretion did not occur before 5 min. These differences in time course paralleled the differences in the onset of translocation of protein kinase C. The results support a direct involvement of protein kinase C in carbamoylcholine-mediated but not in isoproterenol-mediated stimulation of exocytosis in exocrine cells.
Dermatan sulphate proteoglycans from sclera examined by rotary shadowing and electron microscopy
Authors: Ward N P , Scott J E , Cöster L
Published: August 2015

Two dermatan sulphate-containing proteoglycans from bovine sclera were examined by rotary shadowing and electron microscopy, and the results were compared with previous biochemical findings. Both the large iduronate-poor proteoglycan (PGI) and the small iduronate-rich proteoglycan (PGII) possessed a globular proteinaceous region. Whereas PGI had a branched extension from the globular region, with five to eight side chains attached to it, PGII had only a single tail, which was of glycosaminoglycuronan. PGII aggregated via globular-region interactions, which were much diminished by reduction and alkylation. PGI aggregated via side chains and globular-region interactions. Although a few PGI aggregates were large, and similar to the hyaluronan-cartilage proteoglycan aggregates [Weidemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333], hyaluronan did not cause enhanced aggregation. PGII is very similar in shape to the small cartilage chondroitin sulphate proteoglycan, whereas PGI somewhat resembles the large cartilage chondroitin sulphate proteoglycan, although with many fewer glycosaminoglycan side chains, and probably only one globular region as opposed to two in the cartilage proteoglycan.
Purification and partial characterization of a tumour-metastasis-associated high-Mr glycoprotein from rat 13762NF mammary adenocarcinoma cells
Authors: Steck P A , North S M , Nicolson G L
Published: August 2015

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.
Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin
Authors: Seidah N G , Manjunath P , Rochemont J , Sairam M R , Chrétien M
Published: August 2015

Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.
Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa
Authors: Godfrey C , Gadsby P M A , Thomson A J , Greenwood C , Coddington A
Published: August 2015

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.
Inhibition of ferrochelatase during differentiation of murine erythroleukaemia cells
Authors: Fadigan A , Dailey H A
Published: August 2015

During dimethyl sulphoxide-induced differentiation of DS-19 murine erythroleukaemia (MEL) cells, the activity of the terminal enzyme of the haem-biosynthetic pathway, ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), is thought to be the rate-limiting step for haem production. Differentiation of induced MEL cells in the presence of exogeneously supplied protoporphyrin IX showed that total haem production was affected by added porphyrin only after 48 h. These data suggest that iron insertion, the terminal step, is rate-limiting during the first 48 h of differentiation. Addition of low levels of diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine to differentiating cultures resulted in decreased haem production and decreased ferrochelatase activity. N-Methylprotoporphyrin at nanomolar concentrations also strongly inhibited ferrochelatase activity, but had no inhibitory effect on cellular haem production. The bivalent cations Co2+, Cd2+ and Mn2+ were tested for their effect on haem production and ferrochelatase activity. All three metals were found to inhibit both haem formation and ferrochelatase activity, with Mn2+ being the strongest effector. These data, together with those previously published, suggest that the terminal step in haem biosynthesis is rate-limiting during the early stages of differentiation in MEL cells.
Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli
Authors: Roberts A N , Barnett L , Brenner S
Published: August 2015

We report the development of a plasmid-mediated transformation system for Arthrobacter sp. NRRLB3381, using the Streptomyces cloning vector pIJ702. Our procedure gives a transformation frequency of 10(3)/micrograms of plasmid DNA. In addition we have explored the expression of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli, and shown that the ermA promoter is recognized in S. lividans not E. coli. The relationship between Arthrobacter, Streptomyces and E. coli promoters is discussed.
Phospholipid and fatty acid composition in mitochondria from spinach (Spinacia oleracea) leaves and petioles. A comparative study
Authors: Edman K , Ericson I
Published: August 2015

Essentially chlorophyll-free mitochondria from photosynthetic (leaf) and non-photosynthetic tissue (petiole) were isolated from spinach (Spinacia oleracea). Leaf mitochondria were found to contain more phosphatidylcholine than phosphatidylethanolamine compared with petiole mitochondria. Galactolipids were found in small and equal amounts (5 mol of galactolipids/100 mol of galactolipids and phospholipids) in both leaf and petiole mitochondria. Fatty acid composition showed a significant difference in the amounts of C18:2 and C18:3 acids. The C18:2/C18:3 ratio was more than twice as high in all of the phospholipids studied from petiole mitochondria compared with the ratio in leaf mitochondria. More than 50% (mol/100 mol) of the fatty acids in the major lipids (phosphatidylcholine, phosphatidylethanolamine and cardiolipin) in petiole mitochondria were C18:2. In the minor lipids (phosphatidylinositol and phosphatidylglycerol), C16:0 dominated in both leaf and petiole mitochondria.
Cloning and sequencing of the medium-chain S-acyl fatty acid synthetase thioester hydrolase cDNA from rat mammary gland
Authors: Naggert J , Williams B , Cashman D P , Smith S
Published: August 2015

cDNA clones coding for the medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II) from rat mammary gland were identified in a bacteriophage lambda gt11 library and their nucleotide sequences were determined. The predicted coding region spans 263 amino acid residues and includes a sequence identical with that of a peptide derived from the enzyme active site. The rat thioesterase II cDNA sequence exhibits homology with that of a thioesterase found in duck uropygial glands.