bluuebunny/crossref_metadata_2025 huggingface dataset:
l-threonine aldolase is not a genuine enzyme in rat liver
Authors: Yeung Y G
Published: August 2015

Activity of L-threonine aldolase in rat liver cytosolic extract was not affected by the omission of alcohol dehydrogenase in a previously established NADPH-linked alcohol dehydrogenase-coupled assay. The liver extract was able to catalyse the dehydrogenation of NADPH with either acetaldehyde (a product of L-threonine aldolase action) or 2-oxobutyrate (a product of L-threonine dehydratase action). When the liver extract was chromatographed on a Sephacryl S-200 column, no threonine aldolase activity was detected in the eluate. However, activity of threonine aldolase re-appeared when the fractions with highest activity of lactate dehydrogenase and threonine dehydratase were mixed. Activity of threonine aldolase could also be abolished by removing threonine dehydratase from the liver extract with a specific antibody. Hence L-threonine aldolase should not be a genuine enzyme in the rat liver, and the apparent enzyme activity may result from a combined effect of threonine dehydratase and lactate dehydrogenase (or an oxo acid-linked NADPH dehydrogenase) in the liver cytosolic extract.
Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite
Authors: Aviram I , Sharabani M
Published: August 2015

The reduction with dithionite of neutrophil cytochrome b-558, implicated in superoxide generation by activated neutrophils, was investigated by a stopped-flow technique in non-ionic-detergent extracts of the membranes and in crude membrane particles. The dependence of the pseudo-first-order rate constants on the concentration of dithionite was consistent with a mechanism of reduction that involves the dithionite anion monomer SO2.- as the reactive species. The estimated second-order rate constant was 7.8 × 10(6) M-1 × S-1 for Lubrol PX-solubilized cytochrome b-558 and 5.1 × 10(6) M-1 × S-1 for the membrane-bound protein. The similarity of the kinetic constants suggests that solubilization did not introduce gross changes in the reactive site. Imidazole and p-chloromercuribenzoate, known as inhibitors of NADPH oxidase, did not affect significantly cytochrome b-558 reduction rates. The reaction rate of cytochrome b-558 with dithionite exhibited a near-zero activation energy. The first-order rate constant for reduction decreased with increasing ionic strength, indicating a positive effective charge on the reacting protein.
Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulphates
Authors: Hovingh P , Piepkorn M , Linker A
Published: August 2015

We analysed the distribution, structural characteristics, antithrombin-III-binding properties and anticoagulant activities of heparins and heparan sulphates isolated from the tissues of a wide range of vertebrates. Heparin has a curiously limited distribution, since it was absent from lower aquatic vertebrate species, present in only certain organs such as intestine in many higher vertebrates, and completely absent from the rabbit among mammals examined. The heparins were structurally diverse, and they exhibited a broad range of anticoagulant activities, from approx. 50% to 150% of average commercial heparins. Although there was a rough correlation between the anticoagulant potency of the starting isolate and the proportional content of material exhibiting high-affinity binding to the proteinase inhibitor antithrombin III, activities of high-affinity fractions from heparins low in activity overlapped those of low-affinity fractions from highly active heparins. Heparan sulphates, which in contrast were isolated from nearly all vertebrate organs, contained high-affinity subfractions constituting up to 5% of the starting material and possessing anticoagulant potencies of 2-30 units/mg. In consideration of the heparin data, we infer that its biological function is either species-specific or may be served by other molecular elements, and that there exists considerable diversity in the antithrombin-III-binding sequence of heparin. The more-generally distributed glycosaminoglycan heparan sulphate possesses within its variable structure a small high-affinity subfraction with low anticoagulant potency, whether isolated from aorta or other tissues. Although heparan sulphate appears to have an essential function at the cellular level, we suggest that this is probably not that of providing heparin-like antithrombotic effects on vascular surfaces.
The involvement of pyruvate cycling in the metabolism of aspartate and glycerate by the perfused rat kidney
Authors: Scaduto R C , Davis E J
Published: August 2015

The metabolism of glycerate and aspartate was investigated in perfused rat kidneys. The major pathway active for aspartate metabolism and NH3 production was found to include transamination, and not the purine nucleotide cycle. Pyruvate cycling was identified as a means by which reducing potential is generated in the cytosol for glucose and lactate production from these substrates. Inhibition of mitochondrial pyruvate transport caused an inhibition of glucose production, accumulation of lactate and pyruvate in the perfusate, and a decrease in the [lactate]/[pyruvate] ratio in kidneys perfused with aspartate. These data indicate a role of mitochondrial pyruvate transport in the provision of cytosolic reducing potential. With either aspartate or glycerate, 3-mercaptopicolinic acid (3-MPA) suppressed glucose synthesis and caused accumulation of malate plus fumarate within the kidney. Glucose production from glycerate was much less sensitive to the presence of 3-MPA than was glucose production from aspartate, illustrating a phosphoenolpyruvate carboxykinase (PEPCK)-independent pathway for the cycling of pyruvate. In aspartate-perfused kidneys, the presence of 3-MPA, at concentrations that completely blocked glucose accumulation in the perfusate, did not affect the rate of NH3 production and had only a minor effect on the rate of aspartate uptake. These data allow for an estimation of the rate of pyruvate formation from aspartate of about 1 mumol/min per kidney under conditions of complete PEPCK inhibition. Thus a PEPCK-independent pathway is operative for amino acid oxidation and pyruvate formation in perfused kidneys. The NADP-linked, but not the NAD-linked, ‘malic’ enzyme activity of the kidney cortex was found to be sufficient to catalyse this estimated rate of pyruvate formation.
Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice
Authors: Phillips M , Camakaris J , Danks D M
Published: August 2015

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants ‘brindled’ and ‘blotchy’, brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.
Further characterization of the platinum-reactive component of the α2-macroglobulin-receptor recognition site
Authors: Pizzo S V , Roche P A , Feldman S R , Gonias S L
Published: August 2015

alpha 2-Macroglobulin (alpha 2M)-methylamine that had been allowed to react with cis-dichlorodiammineplatinum(II) (cis-DDP) bound with greatly reduced affinity to specific alpha 2M receptors, as determined by macrophage binding studies in vitro and plasma-clearance experiments in vivo. Subsequent reaction with diethyl dithiocarbamate completely restored receptor recognition function. The optimal effect was obtained when the diethyl dithiocarbamate concentration was twice the total platinum concentration. alpha 2M-methylamine that was allowed to react with H2O2 competed less effectively for specific cell-surface binding sites, as demonstrated by studies both in vivo and in vitro. The apparent dissociation constant was increased nearly 7-fold by a 15 min exposure to H2O2. alpha 2M-methylamine was affected significantly less by the H2O2 exposure after pretreatment with cis-DDP. Amino acid analysis indicated that H2O2 treatment of alpha 2M modified 19 of the 25 methionine residues per alpha 2M subunit. Pretreatment with cis-DDP protected two to four of these methionine residues. The only other residue altered by H2O2 treatment of alpha 2M was histidine. A net decrease of two histidine residues per subunit was observed, but cis-DDP pretreatment did not alter this result. In order to rule out the slight possibility that histidine modification might account for the observed H2O2-induced loss in receptor recognition, diethyl pyrocarbonate was employed as a histidine-modifying reagent. This treatment modified 53 histidine residues in both native and fast-form alpha 2M. Fast-form alpha 2M was still recognized by the alpha 2M receptor, as determined by studies both in vivo and in vitro; however, a fraction of the modified protein now cleared via the acyl-low-density-lipoprotein receptor as well. Reaction of diethyl pyrocarbonate-treated alpha 2M with hydroxylamine reversed derivatization of 43 of the 53 histidine residues. Moreover, this treatment also resulted in an alpha 2M fast-form preparation that was recognized only by the alpha 2M receptor. It is concluded that cis-DDP and H2O2 modify a critical methionine residue in the primary sequence of the alpha 2M-receptor recognition site.
Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles
Authors: Bhuvaneswaran C , Mitropoulos K A
Published: August 2015

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.
Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria
Authors: Malloch G D A , Munday L A , Olson M S , Clark J B
Published: August 2015

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.
The relationship between intra- and extra-cellular surfactant phospholipids in the lungs of rabbits and the effects of silica-induced lung injury
Authors: Dethloff L A , Gilmore L B , Hook G E R
Published: August 2015

Extensive homogenization of lung tissue by nitrogen decompression in a Parr disruption bomb increased by 5-fold the yields of low-density phospholipid (d = 1.06) achieved by other methods. This intracellular phospholipid preparation was high in phosphatidylcholines (84.3%), particularly disaturated phosphatidylcholine (51.2%). On the basis of its low density, composition, and morphological appearance, we concluded that this phospholipid was derived from the intracellular compartment of pulmonary surfactant. We examined the relationship between intra- and extra-cellular surfactant pools according to age, gender and silica-induced pulmonary injury. In normal animals the intracellular pool of surfactant phospholipids increased from 1.54 +/- 0.14 mg at 1 day after birth to 62.30 +/- 4.50 mg per pair of lungs after 31 months, and over the same time period the extracellular pool increased from 1.04 +/- 0.15 mg to 27.45 +/- 2.30 mg per pair of lungs. The ratio between the extracellular and intracellular pools of surfactant increased from 1.50 +/- 0.19 at 1 day after birth to 2.28 +/- 0.23 after 31 months of age. The ratio between the two pools was not influenced by gender, but was changed by the intratracheal injection of silica into the lungs. Intratracheal injection of silica dust increased the levels of surfactant in both compartments, but not to the same extent, indicating that the ratio between the pools could be changed by toxic materials. These data suggest the existence of a size relationship between the intra- and the extra-cellular pools of surfactant, a relationship which implies a common regulatory mechanism that can be disturbed during pulmonary injury.
Na+-H+ exchange in luminal-membrane vesicles from rabbit proximal convoluted and straight tubules in response to metabolic acidosis
Authors: Jacobsen C , Kragh-Hansen U , Sheikh M I
Published: August 2015

Na+-H+-exchanger activity of pars convoluta and pars recta luminal-membrane vesicles prepared from the proximal tubule of acidotic and control rabbits were assayed by a rapid-filtration technique and an Acridine Orange method. Both experimental approaches revealed the existence of an antiporter, sensitive to metabolic acidosis, in pars convoluta membrane vesicles. Kinetic data, obtained with the pH-sensitive dye, showed that the Km for Na+ transport was unchanged by acidosis, whereas Vmax. for exchanger activity was increased, on an average, by 44%. The fluorescence method, in contrast with the rapid-filtration technique, was able to detect exchanger activity in pars recta membrane vesicles. The Km value for the antiporter located in pars recta is comparable with that calculated for pars convoluta membrane vesicles. By contrast, the Vmax. of this exchanger is only about 25% of that found for pars convoluta. Furthermore, metabolic acidosis apparently does not increase Na+-H+-exchanger activity of pars recta luminal-membrane vesicles.
Purification and properties of a xylanase from Streptomyces lividans
Authors: Morosoli R , Bertrand J L , Mondou F , Shareck F , Kluepfel D
Published: August 2015

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).
Decreased rate of ketone-body oxidation and decreased activity of d-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats
Authors: Grinblat L , Pacheco Bolaños L F , Stoppani A O
Published: August 2015

Heart mitochondria from chronically diabetic rats (‘diabetic mitochondria’), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats (‘normal mitochondria’). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.
Oxidative radioiodination damage to human lactoferrin
Authors: Rosenmund A , Kuyas C , Haeberli A
Published: August 2015

Oxidative iodination of human lactoferrin (Lf) as commonly performed by using the chloramine-T, the Iodogen or the lactoperoxidase method produces an unreliable tracer protein because of excessive and heterogeneous polymer formation. Before iodination a minor tetramer fraction may be demonstrable in iron-saturated Lf only. Iodination-induced polymerization of iron-poor as well as iron-saturated Lf occurs independently of the presence or absence of 10 mM-EDTA and the 125I-/Lf molar ratio used for iodination. 125I-Lf polymers are mainly covalently linked, as suggested by the lack of substantial dissociation in SDS/polyacrylamide-gel electrophoresis. Damage to the 125I-Lf monomer may be another consequence of oxidative iodination. This is demonstrated in SDS/polyacrylamide-gel electrophoresis where 50% of the radioactivity of apparently normal monomer (Mr 75,000) is displaced to a lower-Mr region (30,000-67,000) after reduction with dithiothreitol. Non-oxidative iodination by the Bolton-Hunter technique produces an antigenetically stable tracer that is not being subjected to polymerization and monomer degradation as judged by high-performance gel chromatography and SDS/polyacrylamide-gel electrophoresis with and without dithiothreitol treatment. It is concluded that oxidation in itself leads to covalent non-disulphide cross-linking between human Lf molecules and, possibly, to intramolecular peptide-bond breaking becoming unmasked under reducing conditions. In biological experiments with human 125I-Lf this problem should be carefully considered.
Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases
Authors: Imai A , Rebecchi M J , Gershengorn M C
Published: August 2015

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.
Erythrocyte nucleoside and sugar transport Endo-β-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins
Authors: Kwong F Y , Baldwin S A , Scudder P R , Jarvis S M , Choy M Y , Young J D
Published: August 2015

Nucleoside- and glucose-transport proteins isolated from human erythrocyte membranes were photoaffinity-labelled with [3H]nitrobenzylthioinosine and [3H]cytochalasin B, respectively, and subjected to endo-beta-galactosidase or endoglycosidase-F digestion. Without enzyme treatment the two radiolabelled transporters migrated on SDS/polyacrylamide gels with the same apparent Mr (average) of 55,000. Apparent Mr (average) values after endo-beta-galactosidase digestion were 47,000 and 48,000 for the nucleoside and glucose transporters respectively, and 44,000 and 45,000 respectively after endoglycosidase-F digestion. In contrast, endo-beta-galactosidase had no effect on the electrophoretic mobility of the nucleoside transporter isolated from pig erythrocytes. This transport system exhibited a higher Mr than the human protein, endoglycosidase-F treatment decreasing its apparent Mr (average) from 64,000 to 57,000. It is concluded that the human and pig erythrocyte nucleoside transporters are glycoproteins containing N-linked oligosaccharide. The data provide evidence of substantial carbohydrate and polypeptide differences between the human and pig erythrocyte nucleoside transporters, but evidence of molecular similarities between the human erythrocyte nucleoside and glucose transporters.
The dependence on Ca2+ of the guanine-nucleotide-activated polyphosphoinositide phosphodiesterase in neutrophil plasma membranes
Authors: Cockcroft S
Published: August 2015

The requirement for Ca2+ for the activation of polyphosphoinositide phosphodiesterase was studied with the guanine nucleotide analogue guanosine 5′-[gamma-thio]triphosphate (GTP gamma S). Levels of Ca2+ that pertain in unstimulated neutrophils (100 nM) are obligatory for the full expression of enzyme activity stimulated with GTP gamma S. Reduction of Ca2+ to 1 nM leads to inhibition. Increasing the level of Ca2+ from 100 nM to 1000 nM does not alter enzyme activity. Guanosine 5′-[beta-thio]diphosphate (GDP beta S) does not stimulate the phosphodiesterase but is an effective inhibitor of activation by GTP gamma S. Ca2+ in the millimolar range can also activate the phosphodiesterase alone and this is not inhibited by GDP beta S. It is also shown that Sr2+ in the millimolar range can stimulate enzyme activity similarly to Ca2+.
Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns
Authors: Cunliffe C J , Franklin T J , Gaskell R M
Published: August 2015

An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays.
Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter
Authors: Anderson S , Smith S M
Published: August 2015

DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast.
The control of CTP:choline-phosphate cytidylyltransferase activity in pea (Pisum sativum L.)
Authors: Price-Jones M J , Harwood J L
Published: August 2015

Several possible control mechanisms for CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity in pea (Pisum sativum L.) stems were investigated. Indol-3-ylacetic acid (IAA) treatment of the pea stems decreased total cytidylyltransferase activity but did not affect its subcellular distribution. Oleate (2 mM) caused some stimulation of enzyme activity by release of activity from the microsomal fraction into the cytosol, but neither phosphatidylglycerol nor monoacyl phosphatidylethanolamine had an effect on activity or subcellular distribution. A decrease in soluble cytidylyltransferase protein concentrations was found in IAA-treated pea stems, but this was not sufficient to account for all of the decrease in cytidylyltransferase activity. A 50% inhibition of enzyme activity could be obtained with 0.2 mM-CMP, which indicated possible allosteric regulation. Similar inhibition was obtained with 1.5 mM-ATP, but other nucleotides had no effect. The cytidylyltransferase enzyme protein was not directly phosphorylated, and the inhibition with 1.5 mM-ATP occurred with the purified enzyme, thus excluding an obligatory mediation via a modulator protein. The results indicate that the cytosolic form of cytidylyltransferase is the most important in pea stem tissue and that the decrease in cytidylyltransferase activity in IAA-treated material appears to be brought about by several methods.
The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa
Authors: Poulos A , Sharp P , Johnson D , White I , Fellenberg A
Published: August 2015

Fatty acids with carbon chain lengths greater than 22 (VLCFA) have been detected in boar, ram, bull and human spermatozoa. Saturated and mono-unsaturated fatty acids were present in all spermatozoa but, except for human spermatozoa, polyenoic fatty acids were quantitatively the most important components. Marked differences in polyenoic fatty acid composition were observed. Whereas human spermatozoa contain predominantly di-, tri- and tetraenoic fatty acids with up to 32 carbon atoms, boar, ram and bull spermatozoa also contain pentaenoic and/or hexaenoic acids with up to 34 carbon atoms. Human and boar spermatozoa differ markedly from those of the ram and bull in that only n-6 series acids are present.
Thiyl free radicals and the oxidation of ferrocytochrome c Direct observation of coupled hydrogen-atom- and electron-transfer reactions
Authors: Forni L G , Willson R L
Published: August 2015

Absolute rate constants for the reaction of ferrocytochrome c with the thiyl radicals derived from cysteine, GSH, penicillamine and N-(2-mercaptopropionyl)glycine were measured by using the technique of pulse radiolysis. The reaction is believed to occur through a one-electron-transfer process, in agreement with the hypothesis that thiols may act as catalysts linking hydrogen-atom- and electron-transfer reactions.
Accumulation of globotriaosylceramide in a case of leiomyosarcoma
Authors: Li S C , Kundu S K , Degasperi R , Li Y T
Published: August 2015

Analysis of the glycosphingolipid composition in one case of uterine leiomyosarcoma metastasized to the liver showed an accumulation of globotriaosylceramide as compared with normal liver and uterus from which the tumour originated. The structure and the amount of glycosphingolipids were established by using specific glycosidases, permethylation analysis and h.p.l.c. The reason for the accumulation of globotriaosylceramide in leiomyosarcoma remains to be answered.
Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C
Authors: Ikehara Y , Hayashi Y , Ogata S , Miki A , Kominami T
Published: August 2015

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.
Differential effects of acute changes in cell Ca2+ concentration on myofibrillar and non-myofibrillar protein breakdown in the rat extensor digitorum longus muscle in vitro. Assessment by production of tyrosine and Nτ-methylhistidine
Authors: Goodman M N
Published: August 2015

The influence of Ca2+ on myofibrillar proteolysis was evaluated in the isolated extensor digitorum longus muscle incubated in vitro with agents previously shown to increase the intracellular concentration of Ca2+. Myofibrillar proteolysis was evaluated by measuring the release of N tau-methylhistidine, and total proteolysis was evaluated by measuring tyrosine release by incubated muscles after the inhibition of protein synthesis with cycloheximide. Incubated muscles released measurable quantities of N tau-methylhistidine, and muscle contents of the amino acids remained stable over 2 h of incubation. The release of N tau-methylhistidine by incubated muscles was similar to its release by perfused rat muscle in response to brief starvation, indicating the integrity of the incubated muscles. Ca2+ ionophore A23187, dibucaine, procaine, caffeine and elevated K+ concentration increased lactate release by incubated muscles and decreased tissue contents of ATP and phosphocreatine to varying degrees, indicating the metabolic effectiveness of the agents tested. Only A23187 and dibucaine increased total cell Ca2+, and they increased tyrosine release. Caffeine and elevated [K+] increased neither cell Ca2+ nor tyrosine release; however, only A23187 and dibucaine increased tyrosine release significantly. On the other hand, these agents were without effect on myofibrillar proteolysis as assessed by N tau-methylhistidine release by incubated muscles and changes in tissue contents of the amino acid. In fact, some of the agents tested tended to decrease myofibrillar proteolysis slightly. These results indicate that acute elevation of intracellular Ca2+ is associated with increased breakdown of non-myofibrillar but not myofibrillar proteins. Because of this, the role of elevated Ca2+ in muscle atrophy in certain pathological states is questioned. The data also indicate that the breakdown of myofibrillar and non-myofibrillar proteins in muscle is regulated independently and by different pathways, a conclusion reached in previous studies with perfused rat muscle.
Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity
Authors: Spaltro J , Alhadeff J A
Published: August 2015

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.
Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells
Authors: Vischer U , Blondel B , Wollheim C B , Höppner W , Seitz H J , Iynedjian P B
Published: August 2015

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.
Molecular biology and immunology of cytomegalovirus
Authors: Griffiths P D , Grundy J E
Published: August 2015

The application of modern biochemical techniques has led to a rapid improvement in our knowledge of the molecular biology of CMV. Several coding regions of the DNA genome have been identified with certainty and major virus-coded proteins have been given provisional names. The cascade expression of the CMV genome has been shown to be controlled by mechanisms similar to those found in other herpes viruses, together with novel post-transcriptional controls which remain to be defined. The control of CMV replication by the host involves both non-specific and specific defence mechanisms. The induction of natural killer cells and interferon early after CMV infection appears to be the most important aspects of the non-specific host defence against the virus. The cell-mediated immune response, in particular the generation of Tc cells against CMV early antigens, is probably the most important facet of the specific immune defence against CMV. When intact these defence mechanisms appear to be efficient in restricting viral replication; however, when such immunity is compromised, the balance rapidly swings in favour of the virus. As our understanding of the interaction between the host and the virus increases, it may be possible to redress the balance in such cases in favour of the host.
Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen
Authors: Jenö P , Green J R , Lentze M J
Published: August 2015

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.
Short-term stimulation of Na+-dependent amino acid transport by dibutyryl cyclic AMP in hepatocytes. Characteristics and partial mechanism
Authors: Moule S K , Bradford N M , McGivan J D
Published: August 2015

The short-term protein-synthesis-independent stimulation of alanine transport in hepatocytes was further investigated. Cyclic AMP increased the Vmax. of alanine transport. Amino acid transport via systems A, ASC and N was stimulated. A good correlation was found between the initial rate of transport and the cell membrane potential as calculated from the distribution of Cl-. Cyclic AMP increased the rate of alanine transport, stimulated Na+/K+ ATPase (Na+/K+-transporting ATPase) activity and caused membrane hyperpolarization. The time courses and cyclic AMP dose-dependencies of all three effects were similar. Ouabain abolished the effect of cyclic AMP on Cl- distribution and on transport of alanine. The effect of cyclic AMP on alanine transport and Cl- distribution was mimicked by the antibiotic nigericin; the effect of nigericin was also abolished by ouabain. It is concluded that the effect of cyclic AMP on transport is mediated via membrane hyperpolarization. It is suggested that the primary action of cyclic AMP is to increase the activity of an electroneutral Na+/K+-exchange system in the liver cell plasma membrane, thus hyperpolarizing the membrane by stimulating the electrogenic Na+/K+ ATPase.
Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes
Authors: Whipps D E , Armston A E , Pryor H J , Halestrap A P
Published: August 2015

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.