bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidylinositol 4,5-bisphosphate is selectively retained by platelet-fibrin clots formed by thrombin
Authors: Vickers J D , Kinlough-Rathbone R L , Mustard J F
Published: August 2015

Stimulation of human or rabbit platelets with thrombin in the presence of fibrinogen caused a large decrease, compared with unstimulated controls, in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2) that could be extracted with acidified chloroform/methanol (60% at 60 s). In contrast, stimulation in the absence of added fibrinogen increased the amount of PIP2. The decrease was specific for PIP2, because similar decreases could not be demonstrated for other phosphoinositides or phospholipids. The interaction of polymerizing fibrin with stimulated platelets was required for the decrease in PIP2, since polymerized fibrin formed by reptilase did not cause the decrease in the amount of extractable PIP2, and inhibition by glycyl-L-prolyl-L-arginyl-L-proline of polymerization of fibrin formed by the action of thrombin prevented the large decrease in extractable PIP2. The decrease in extractable PIP2 could not be explained by increased degradation of PIP2, since sufficient degradation products were not formed. Thus, when platelets are stimulated with thrombin in the presence of fibrinogen, an association of polymerizing fibrin with the stimulated platelets occurs that leads to decreased extractability of PIP2. This may mean that PIP2 forms a specific association with platelet proteins that are involved in clot retraction.
cDNA clones encoding rabbit immunoglobulin λ chains. Evidence for length variation of the third hypervariable region and for a novel constant region
Authors: Hayzer D J , Duvoisin R M , Jaton J C
Published: August 2015

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.
Effects of diet on the function of sarcoplasmic reticulum
Authors: Gould G W , McWhirter J M , East J M , Lee A G
Published: August 2015

We have examined the effect of diet on the phospholipid composition of the sarcoplasmic reticulum of rabbit muscle. Enriching the diet with corn or fish oil results in significant changes in the fatty acyl chain composition of the various phospholipid classes, with relatively little change in the relative contents of the phospholipids. These alterations in composition have no significant effect on the ATPase activity of vesicles of sarcoplasmic reticulum or on the pattern of Ca2+ uptake and release.
Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-glucose
Authors: Ludwigs U , Elgavish A , Esko J D , Meezan E , Rodén L
Published: August 2015

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.
Amino acid sequence of acyl-CoA-binding protein from cow liver
Authors: Mikkelsen J , Højrup P , Nielsen P F , Roepstorff P , Knudsen J
Published: August 2015

Acyl-CoA-binding protein from bovine liver was purified with the use of reverse-phase h.p.l.c. in the final step. The complete amino acid sequence was determined by using a combination of gas-phase Edman degradation and electron-impact and fast-atom-bombardment mass spectrometry. The sequence was confirmed by determination of the Mr by plasma-desorption time-of-flight mass spectrometry.
A 31P-n.m.r. study of the acute effects of β-blockade on the bioenergetics of skeletal muscle during contraction
Authors: Challiss R A , Hayes D J , Radda G K
Published: August 2015

1. The effects of beta-adrenoceptor antagonist administration on skeletal muscle contractile performance and bioenergetics in vivo have been investigated during unilateral sciatic nerve stimulation in the rat. 2. Two muscle stimulation protocols have been used: supramaximal stimulation at 4 Hz, or incremental supramaximal stimulation at 1, 2 and 4 Hz. Changes in high-energy phosphate concentrations were followed using 31P-n.m.r., and gastrocnemius muscle twitch characteristics were monitored continuously. 3. Under all conditions investigated, DL-propranolol administration (2.5 mg/kg body wt.) caused a significant decrease in cyclic AMP concentrations in resting and stimulated gastrocnemius muscle, prevented an increase in heart rate upon muscle stimulation, but did not affect plasma glucose, fatty acid or lactate concentrations in comparison with values obtained in control experiments. 4. Administration of DL-propranolol 5 min or 35 min before unilateral stimulation of 4 Hz had no effect on changes in muscle phosphocreatine, ATP or Pi concentrations, intracellular pH or contractile performance. 5. In contrast, animals receiving DL-propranolol 5 min before unilateral stimulation of 1, 2 and 4 Hz showed a significant deterioration in gastrocnemius muscle tension development during 2 and 4 Hz stimulation compared with control animals. Concurrent with this change in contractile performance was a higher muscle concentration of phosphocreatine, a lower concentration of Pi and no significant change in intramuscular pH compared with control experiments. 6. The changes in muscle performance and bioenergetics observed during the incremental stimulation protocol were not observed when D-propranolol was administered and could be completely circumvented by a short period of muscle stimulation of 4 Hz prior to initiation of the incremental stimulation protocol. 7. Mechanisms are discussed which may account for the failure of gastrocnemius muscle to generate the expected force during the incremental stimulation protocol in the presence of beta-blockade.
Purification and characterization of glutathione S-transferases of human kidney
Authors: Singh S V , Leal T , Ansari G A , Awasthi Y C
Published: August 2015

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (α, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST α, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.
Effects of fasting on tissue glucose utilization in conscious resting rats. Major glucose-sparing effect in working muscles
Authors: Issad T , Pénicaud L , Ferré P , Kandé J , Baudon M A , Girard J
Published: August 2015

The effects of fasting on glucose metabolism in the conscious resting rat were studied. Fasting decreased whole-body glucose utilization by 40%. The fast induced a decrease in glucose utilization in muscles which are constantly working even in the resting state, i.e. heart, diaphragm and postural muscles. No modification was observed in other tissues.
Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties
Authors: Freeman C , Hopwood J J
Published: August 2015

Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.
Cell-free acylation of rat brain myelin proteolipid protein and DM-20
Authors: Yoshimura T , Agrawal D , Agrawal H C
Published: August 2015

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of ‘PLP’ [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and ‘DM-20’ [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.
Analysis of tricarboxylic acid-cycle metabolism of hepatoma cells by comparison of 14CO2 ratios
Authors: Kelleher J K , Bryan B M , Mallet R T , Holleran A L , Murphy A N , Fiskum G
Published: August 2015

The CO2-ratios method is applied to the analysis of abnormalities of TCA (tricarboxylic acid)-cycle metabolism in AS-30D rat ascites-hepatoma cells. This method utilizes steady-state 14CO2-production rates from pairs of tracers of the same compound to evaluate TCA-cycle flux patterns. Equations are presented that quantitatively convert CO2 ratios into estimates of probability of flux through TCA-cycle-related pathways. Results of this study indicated that the ratio of 14CO2 produced from [1,4-14C]succinate to 14CO2 produced from [2,3-14C]succinate was increased by the addition of glutamine (5 mM) to the medium. An increase in the succinate CO2 ratio is quantitatively related to an increased flux of unlabelled carbon into the TCA-cycle-intermediate pools. Analysis of 14C distribution in [14C]citrate derived from [2,3-14C]succinate indicated that flux from the TCA cycle to the acetyl-CoA-derived carbons of citrate was insignificant. Thus the increased succinate CO2 ratio observed in the presence of glutamine could only result from an increased flux of carbon into the span of the TCA cycle from citrate to oxaloacetate. This result is consistent with increased flux of glutamine to alpha-oxoglutarate in the incubation medium containing exogenous glutamine. Comparison of the pyruvate CO2 ratio, steady-state 14CO2 production from [2-14C]pyruvate versus [3-14C]pyruvate, with the succinate 14CO2 ratio detected flux of pyruvate to C4 TCA-cycle intermediates in the medium containing glutamine. This result was consistent with the observation that [14C]aspartate derived from [2-14C]pyruvate was labelled in C-2 and C-3. 14C analysis also produced evidence for flux of TCA-cycle carbon to alanine. This study demonstrates that the CO2-ratios method is applicable in the analysis of the metabolic properties of AS-30D cells. This methodology has verified that the atypical TCA-cycle metabolism previously described for AS-30D-cell mitochondria occurs in intact AS-30D rat hepatoma cells.
Developmental regulation of rat lung Cu,Zn-superoxide dismutase
Authors: Hass M A , Massaro D
Published: August 2015

In the present investigation we found that lung Cu, Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu, Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu, Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu, Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu, Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu, Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu, Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.
Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin
Authors: Troen B R , Gal S , Gottesman M M
Published: August 2015

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3′ non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.
The trans-stilbene oxide-active glutathione transferase in human mononuclear leucocytes is identical with the hepatic glutathione transferase μ
Authors: Seidegård J , Guthenberg C , Pero R W , Mannervik B
Published: August 2015

A glutathione transferase from human mononuclear leucocytes with high activity towards trans-stilbene oxide (GT-tSBO) was purified. GT-tSBO is expressed in only about 50% of the individuals studied. As judged from activity measurements, immunological studies and the fact that only those individuals who express glutathione transferase mu have high activity towards trans-stilbene oxide, it is concluded that the hepatic transferase mu is identical with the glutathione transferase (GT-tSBO) in mononuclear leucocytes.
Inactivation of the thiol RTEM-1 β-lactamase by 6-β-bromopenicillanic acid. Identity of the primary active-site nucleophile
Authors: Knap A K , Pratt R F
Published: August 2015

The thiol RTEM-1 beta-lactamase [Sigal, Harwood & Arentzen (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme. Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate. Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group. These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover. The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm. Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently. Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase.
Pig kidney angiotensin converting enzyme. Purification and characterization of amphipathic and hydrophilic forms of the enzyme establishes C-terminal anchorage to the plasma membrane
Authors: Hooper N M , Keen J , Pappin D J C , Turner A J
Published: August 2015

Angiotensin converting enzyme from pig kidney was isolated by affinity chromatography after solubilization from the membrane by one of four different procedures. Solubilization with Triton X-100, trypsin or by an endogenous activity in microvillar membranes all generated hydrophilic forms of the enzyme as assessed by phase separation in Triton X-114 and failure to incorporate into liposomes. Only when solubilization and purification was effected by Triton X-100 in the presence of EDTA (10 mM) could an amphipathic form of the enzyme (membrane- or m-form) be generated. The m-form of angiotensin converting enzyme (ACE) appeared slightly larger (Mr approx. 180,000) than the hydrophilic forms (Mr approx. 175,000) after SDS/polyacrylamide-gel electrophoresis, and the m-form incorporated into liposomes, consistent with retention of the membrane anchor. The m-form of ACE showed an N-terminal sequence identical with that of preparations of enzyme isolated after solubilization with detergent alone (d-form), with trypsin (t-form) or by the endogenous mechanism (e-form). These data imply that ACE is anchored to the plasma membrane via its C-terminus, in contrast with the N-terminal anchorage of endopeptidase-24.11. No release of ACE from the membrane could be detected with a variety of phospholipases, including bacterial phosphatidylinositol-specific phospholipases C, although an endogenous EDTA-sensitive membrane-associated hydrolase was capable of releasing a soluble, hydrophilic, form of the enzyme.
Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation
Authors: Whitman M , Kaplan D , Roberts T , Cantley L
Published: August 2015

Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.
Expression of the cDNA encoding for mouse sperm-specific lactate dehydrogenase C in Chinese-hamster ovary cells
Authors: Hou E W , Sakai I , Li S S L
Published: August 2015

The cloned cDNA encoding for mouse sperm-specific lactate dehydrogenase C (LDH-C) was inserted immediately downstream to the MMTV 5′ LTR promoter, and it was shown to synthesize mouse LDH-C polypeptide in Chinese-hamster ovary cells. The mouse LDH-C subunit and the endogenous Chinese-hamster LDH-A subunit formed in vivo a heterotetrameric LDH-A3C1 isoenzyme, and this novel isoenzyme exhibited enzymic activity utilizing lactate as substrate.
Short-term effect of a high-protein/low-carbohydrate diet on aminopeptidase in adult rat jejunoileum. Site of aminopeptidase response
Authors: Raul F , Goda T , Gossé F , Koldovský O
Published: August 2015

The short-term effects of high-protein/low-carbohydrate diet on aminopeptidase N activity were studied in the brush-border membranes of proximal jejunum and proximal ileum of adult rats. The animals were starved overnight and re-fed for 15 h either with a standard diet (20% protein, 55% carbohydrate, in terms of energy content) or with a high-protein/low-carbohydrate diet of equal energy content (70% protein, 5% carbohydrate). All rats consumed similar amounts of diet, and measurements were made 15 h after initiation of re-feeding. In the proximal jejunum a slight increase in aminopeptidase activity was observed after the high-protein intake. In contrast, considerable stimulation (52%) of the enzyme specific activity was obtained in the proximal ileum. This increase in ileal aminopeptidase activity was more prominent in the mature cells of the upper villus. To determine if the increase of aminopeptidase activity was due to an increased amount of enzyme protein, rocket immunoelectrophoresis was performed with detergent-solubilized brush-border protein from ileum on agarose gels containing anti-(rat brush-border) antiserum. When the same amount of enzyme activity was loaded on the gels, the peaks of immunoprecipitate for aminopeptidase were similar for animals fed on a standard or a high-protein diet. When the same amount of protein was loaded, the peak of immunoprecipitate for aminopeptidase was higher (81%) after a high-protein diet. These results showed that the high protein intake evoked an increase in aminopeptidase activity, with a concomitant increase in the amount of immunoreactive protein.
C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells
Authors: Gargosky S E , Wallace J C , Upton F M , Ballard F J
Published: August 2015

1. Synthetic peptides corresponding to the five, seven, nine and eleven C-terminal amino acids of the tetradecapeptide bombesin as well as bombesin itself and gastrin-releasing peptide have been evaluated in Swiss 3T3 cells in order to define the minimal peptide length needed for biological responsiveness. 2. Gastrin-releasing peptide, bombesin, the undecapeptide and nonapeptide had nearly equipotent abilities to compete for binding of labelled gastrin-releasing peptide to the cell receptors and showed half-maximal competition at 5-10 nM. The heptapeptide and pentapeptide were ineffective. 3. Cross-linking experiments demonstrated specific binding of gastrin-releasing peptide to a 100 kDa receptor subunit. 4. Total cell protein synthesis was stimulated equally by the nonapeptide and longer peptides with a half-maximal effect at 0.5 nM, while a more than 1000-fold higher concentration of the heptapeptide was required to produce a similar response. Comparable results were found when insulin was also present. 5. Neither an inhibition of protein breakdown nor a stimulation of DNA labelling could be demonstrated by bombesin or gastrin-releasing peptide. 6. We conclude that a C-terminal peptide ligand comprising more than seven but no more than nine amino acids is required to achieve high-affinity binding and receptor-mediated responses via the bombesin receptor.
Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing
Authors: Hughes R C , Foddy L , Bause E
Published: August 2015

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.
Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane
Authors: Ashby G A , Dilworth M J , Thorneley R N F
Published: August 2015

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.
Primary structure of the glycans from mouse serum and milk transferrins
Authors: Leclercq Y , Sawatzki G , Wieruszeski J M , Montreuil J , Spik G
Published: August 2015

A ‘serotransferrin-like’ protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk ‘serotransferrin-like’ protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk ‘serotransferrin-like’ protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk ‘serotransferrin-like’ protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).
The dephosphorylation pathway of d-myo-inositol 1,3,4,5-tetrakisphosphate in rat brain
Authors: Erneux C , Delvaux A , Moreau C , Dumont J E
Published: August 2015

Dephosphorylation of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was measured in both the soluble and the particulate fractions of rat brain homogenates. Analysis of the hydrolysis of [4,5-32P]Ins(1,3,4,5)P4 showed that for both fractions the 5-phosphate of Ins(1,3,4,5)P4 was removed and inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] was specifically produced. In the soluble fraction, Ins(1,3,4)P3 was further hydrolysed at the 1-phosphate position to inositol 3,4-bisphosphate[Ins(3,4)P2]. DEAE-cellulose chromatography of the soluble fraction separated the phosphatase activities into three peaks. The first hydrolysed both Ins(1,3,4,5)P4 and inositol 1,4,5-trisphosphate, the second inositol 1-phosphate and the third Ins(1,3,4)P3 and inositol 1,4-bisphosphate, [Ins(1,4)P2]. Further purification of the third peak on either Sephacryl S-200 or Blue Sepharose could not dissociate these two activities [i.e. with Ins(1,4)P2 and Ins(1,3,4)P3 as substrates]. The dephosphorylation of Ins(1,3,4)P3 could be inhibited by the addition of Li+.
Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein)
Authors: Yoshida H , Inagaki T , Hirano M , Sugimoto T
Published: August 2015

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a ‘conjugated’ form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.
Absence of endo-β-N-acetylglucosaminidase activity in the kidneys of sheep, cattle and pig
Authors: Song Z W , Li S C , Li Y T
Published: August 2015

The kidneys of man, sheep, cattle and pig were all found to contain 1-aspartamido-beta-acetylglucosamine amidohydrolase activity. However, among these, only human kidney was found to contain endo-beta-N-acetylglucosaminidase activity. The absence of this enzyme in the kidneys of sheep and cattle explains why the oligosaccharides accumulated in, and excreted by, sheep and cattle afflicted with disorders of glycoprotein catabolism (i.e. alpha-mannosidosis and beta-mannosidosis) contain two N-acetylglucosamine residues at the reducing terminus instead of one, as is the case for human patients afflicted with similar disorders.
Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells
Authors: Slotte J P , Bierman E L
Published: August 2015

The spontaneous turnover of plasma-membrane sterols, as measured by their transfer to the endoplasmic reticulum, was measured in quiescent cultured human skin fibroblasts and monkey arterial smooth-muscle cells. The plasma-membrane sterol pool was pulse-labelled with trace amounts of either [3H]desmosterol or [3H]cholesterol. We then measured the enzymic conversion of [3H]desmosterol into [3H]cholesterol and of [3H]cholesterol into [3H]cholesteryl esters in intact cells. Depending on the probe used, markedly different transfer or conversion rates were found in these cells. In quiescent human skin fibroblasts, incubated in a serum-free medium, about 1.1% of the plasma-membrane [3H]desmosterol was converted into [3H]cholesterol/h, whereas in monkey arterial smooth-muscle cells the corresponding rate was 0.4%. Under similar experimental conditions, these cells esterified less than 0.02% (fibroblasts) and 0.12% (smooth-muscle cells) of the plasma-membrane [3H]cholesterol/h. The movement of sterols from the plasma membrane to the endoplasmic reticulum, as measured by the conversion of [3H]desmosterol into [3H]cholesterol was not blocked by colchicine, but was markedly enhanced by 3% (w/v) dimethyl sulphoxide. In all, these results indicate that plasma-membrane sterols of cultured cells are continuously transferred to the interior of the cell at a rate substantially higher than previously appreciated. This turnover of plasma-membrane sterol molecules took place even when there was no mass transfer of sterols into the cells.
Absence of direct coenzyme transfer in an A-B dehydrogenase system
Authors: Ehrlich R S
Published: August 2015

Direct transfer of NADPH between two NADP-dependent dehydrogenases, isocitrate dehydrogenase and glutamate dehydrogenase, has been investigated. These enzymes have opposite stereospecificity for hydrogen transfer to the coenzyme. In contrast with the general direct-transfer mechanism postulated for NAD-dependent dehydrogenases [Srivastava & Bernhard (1986) Science 234, 1081-1086], no evidence for direct transfer in either direction was found for these NADP-dependent dehydrogenases.
The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme
Authors: Plat H , Krenn B E , Wever R
Published: August 2015

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.
Amino acid sequences of cytochrome c-554(548) and cytochrome c' from a halophilic denitrifying bacterium of the genus Paracoccus
Authors: Ambler R P , Daniel M , McLellan L , Meyer T E , Cusanovich M A , Kamen M D
Published: August 2015

The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M. 203 (= A.T.C.C. 12084, N.C.I.B. 8669) have been determined. Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly. The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris. Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence. Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c. Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K. from which copies are available on prepayment.
Reagents for reversible coupling of proteins to the active centres of trypsin-like serine proteinases
Authors: Kalindjian S B , Smith R A G
Published: August 2015

In order to extend the scope for the application of acyl-enzymes as fibrinolytic agents, p-amidinophenyl ester enzyme substrates were prepared that gave stabilized acyl-enzymes capable of being coupled to other proteins. Coupling was achieved by reaction of protein thiol functions with a 2-pyridyldithio moiety within the acyl group. Acyl-enzymes derived from such substrates were stable enough to permit isolation of reversible conjugates between proteins and the active centres of plasminogen activators. Hydrolytic release of active enzyme from a conjugate of human immunoglobulin G and urokinase could be demonstrated.
Thrombin and C-kinase activators potentiate calcium-stimulated arachidonic acid release in human platelets
Authors: Halenda S P , Rehm A G
Published: August 2015

Human platelets were depleted of intracellular Ca2+ and then made selectively permeable to external Ca2+ by addition of the ionophore ionomycin. In this cell system a rapid release of arachidonic acid was seen in direct response to added Ca2+ at concentrations corresponding to cytosolic Ca2+ levels measured in thrombin-stimulated platelets. Thrombin and other activators of Ca2+/phospholipid-dependent protein kinase (C-kinase) potentiated the Ca2+-stimulated arachidonic acid release while exerting little or no effect in the absence of added Ca2+. Agents which increase (R59022) or decrease (isoquinolinesulphonylmethylpiperazine) the activation of C-kinase correspondingly enhanced or inhibited, respectively, the potentiation of arachidonic acid release caused by thrombin. These results support the hypothesis that arachidonic acid release in human platelets is regulated by a co-operative action between intracellular Ca2+ and C-kinase.
The role of C-4-substituted mannose analogues in protein glycosylation. Effect of the guanosine diphosphate esters of 4-deoxy-4-fluoro-d-mannose and 4-deoxy-d-mannose on lipid-linked oligosaccharide assembly
Authors: McDowell W , Grier T J , Rasmussen J R , Schwarz R T
Published: August 2015

The effects of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose (GDP-4FMan) and 4-deoxy-D-mannose (GDP-4dMan) on reactions of the dolichol pathway in chick-embryo cell microsomal membranes were investigated by studies with chick-embryo cell microsomal membranes in vitro and in baby-hamster kidney (BHK) cells in vivo. Each nucleotide sugar analogue inhibited lipid-linked oligosaccharide biosynthesis in a concentration-dependent manner. GDP-4FMan blocked in vitro the addition of mannose to Dol-PP-(GlcNAc)2Man from GDP-Man (where Dol represents dolichol), but did not interfere with the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2. Although GDP-4FMan and Dol-P-4FMan were identified as metabolites of 4FMan in BHK cells labelled with [1-14C]4FMan, GDP-4FMan was a very poor substrate for GDP-Man: Dol-P mannosyltransferase and Dol-P-4FMan could only be synthesized in vitro if the chick-embryo cell membranes were primed with Dol-P. It therefore appears that the inhibition of lipid-linked oligosaccharide formation in BHK cells treated with 4FMan [Grier & Rasmussen (1984) J. Biol. Chem. 259, 1027-1030] is due primarily to a blockage in the formation of Dol-PP-(GlcNAc)2Man2 by GDP-4FMan. In contrast, GDP-4dMan was a substrate for those mannosyltransferases that catalyse the transfer of the first five mannose residues to Dol-PP-(GlcNAc)2. In addition, GDP-4dMan was a substrate for GDP-Man: Dol-P mannosyltransferase, which catalysed the formation of Dol-P-4dMan. As a consequence of this, the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2 may be inhibited through competition for Dol-P. In BHK cells treated with 10 mM-4dMan, Dol-PP-(GlcNAc)2Man9 was the major lipid-linked oligosaccharide detected. Nearly normal extents of protein glycosylation were observed, but very little processing to complex oligosaccharides occurred, and the high-mannose structures were smaller than in untreated cells.
Renal transport of neutral amino acids. Cation-dependent uptake of l-alanine by luminal-membrane vesicles
Authors: Jørgensen K E , Sheikh M I
Published: August 2015

The characteristics of L-alanine transport in luminal-membrane vesicles isolated either from whole cortex or from pars convoluta or pars recta of rabbit proximal tubules were studied by a rapid filtration technique and by a spectrophotometric method. Uptake of L-alanine by vesicles from whole cortex was mediated by both Na+-dependent and Na+-independent, but electrogenic, processes. The nature, mechanism and tubular localization of the transport systems were studied by the use of vesicles derived from pars convoluta and pars recta. In vesicles from pars recta transport of L-alanine was strictly dependent on Na+ and occurred via a dual transport system, namely a high-affinity (half-saturation 0.14 mM) and a low-affinity system (half-saturation 9.6 mM). The cation-dependent but Na+-unspecific transport system for L-alanine was exclusively localized to the pars convoluta, which also contained an Na+-preferring system of intermediate affinity (half saturation 2.1 mM). A closer examination of the mechanism of transport of L-alanine in vesicles from pars convoluta revealed that an H+ gradient (extravesicular greater than intravesicular) can drive the transport of L-alanine into the vesicles both in the presence and in the absence of Na+. The physiological importance of various L-alanine transporters is briefly discussed.