bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of lactate on pancreatic islets. Lactate efflux as a possible determinant of islet-cell depolarization by glucose
Authors: Best L , Yates A P , Meats J E , Tomlinson S
Published: August 2015

The secretion of insulin from perifused rat pancreatic islets was stimulated by raising the glucose concentration from 5.6 to 20 mM or by exposure to tolbutamide. The addition of sodium lactate (40 mM) to islets perifused in the presence of glucose (5.6 mM) resulted in a small, transient, rise in the rate of secretion. The subsequent removal of lactate, but not glucose or tolbutamide, from the perifusate produced a dramatic potentiation of insulin release. The rate of efflux of 45Ca2+ was also increased when islets were exposed to a high concentration of glucose or lactate or to tolbutamide, and again subsequently upon withdrawal of lactate. Efflux of 86Rb+ was modestly inhibited upon addition of lactate and markedly enhanced by the subsequent withdrawal of lactate from islets. The output of [14C]lactate from islets incubated in the presence of [U-14C]glucose increased linearly with increasing concentrations of glucose (1-25 mM). It is proposed that the activation of islets by the addition or withdrawal of lactate is not due to increased oxidative flux, but occurs as a result of the electrogenic passage of lactate ions across the plasma membrane, resulting in islet-cell depolarization, Ca2+ entry and insulin secretion. The production of lactate via the glycolytic pathway, and the subsequent efflux of lactate from the islet cells with concomitant exchange of H+ for Na+, could be a major determinant of depolarization and hence insulin secretion, in response to glucose.
Purification of two glycoproteins expressing β1-6 branched Asn-linked oligosaccharides from metastatic tumour cells
Authors: Laferte S , Dennis J W
Published: August 2015

Increased branching at the trimannosyl core of ‘complex-type’ Asn-linked oligosaccharides has been observed in both human and murine tumour cells, and appears to be associated with enhanced metastatic potential in several murine tumour models [Dennis, Laferte, Waghorne, Breitman & Kerbel (1987), Science 236, 582-585]. The lectin leucoagglutinin (L-PHA) requires the-GlcNAc beta 1-6Man alpha 1-6Man-linked lactosamine antenna in complex-type oligosaccharides for high-affinity binding and can be used to detect these structures in glycoproteins separated on SDS/polyacrylamide-gel electrophoresis. The major L-PHA-binding glycoproteins in the highly metastatic lymphoid tumour cell line called MDAY-D2 were purified and resolved into two major species, termed P2A (110 kDa) and P2B (130 kDa). P2A had L-PHA-reactive Asn-linked oligosaccharides with polylactosamine sequences as well as a large component of sialylated O-linked carbohydrates. The glycoprotein showed structural characteristics similar to those of leukosialin (i.e. CD43), a glycoprotein previously identified on the surface of leukocytes. Based on monosaccharide compositional analysis and glycosidase digestions, P2B was found to be 50-60% Asn-linked oligosaccharide containing polylactosamine sequences and sialic acid. The N-terminal peptide sequence of P2B was determined to be very similar to that of murine lysosomal membrane glycoprotein (LAMP-1), a ubiquitous glycoprotein found largely in the lysosomal membranes but also in the plasma membrane of several murine and human tumour cell lines.
Kinetic evidence for a two-step mechanism for the binding of chymotrysin to α1-proteinase inhibitor
Authors: Bruch M , Bieth J G
Published: August 2015

We have used the proflavin displacement method and a stopped-flow apparatus to measure the rate constant for the binding of 2 microM-chymotrypsin to 20-125 microM-alpha 1-proteinase inhibitor. The observed pseudo-first-order constant showed a hyperbolic dependence on alpha 1-proteinase inhibitor concentration, suggesting a reaction mechanism in which a fast pre-equilibrium (K = 0.19 mM) is followed by a first-order formation of the final complex (k = 252 s-1).
Inositol 1,3,4,5-tetrakisphosphate causes release of Ca2+ from permeabilized mouse lymphoma L1210 cells by its conversion into inositol 1,4,5-trisphosphate
Authors: Cullen P J , Irvine R F , Drøbak B K , Dawson A P
Published: August 2015

Addition of Ins(1,3,4,5)P4 at micromolar concentrations causes release of Ca2+ from electroporated L1210 cells, but not from digitonin-permeabilized cells. This was shown to be due to its conversion into Ins(1,4,5)P3, because only the electroporated cells convert Ins(1,3,4,5)P4 into Ins(1,4,5)P3. Thus electroporation appears to activate or expose an Ins(1,3,4,5)P4 3-phosphatase.
Mammalian protein methylesterase. Physical and enzymic properties
Authors: Veeraragavan K , Gagnon C
Published: August 2015

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.
Luminescence emission from Neurospora copper metallothionein. Time-resolved studies
Authors: Beltramini M , Giacometti G M , Salvato B , Giacometti G , Münger K , Lerch K
Published: August 2015

The luminescence lifetime of Cu-metallothionein from the fungus Neurospora crassa has been studied by the frequency-domain emission technique. Lifetimes of 10.3 and 3.4 microseconds have been found for the protein in the absence and in the presence of oxygen respectively. Binding of Hg(II) results in a quenching of the luminescence correlated to the shortening of lifetime to 0.3-0.4 microsecond. No quenching by oxygen is found for the Hg(II)-Cu-metallothionein adduct. By analogy to model compounds, luminescence emission is attributed to a triplet excited state of a Cu(I)-to-sulphur charge-transfer complex. The comparison of bimolecular quenching constants for O2 and acrylamide indicates a highly compact structure of the protein.
Application of the synthetic method to other amines
Authors: Sparkes M J , Dixon H B F , Geoghegan K F , Visedo-Gonzalez E
Published: August 2015

Taurine and 2-aminoethylphosphonic acid were synthesized by the method of the main paper [Geoghegan & Dixon (1989) Biochem. J. 260, 295-296], i.e. by treating the corresponding halo compound with 2-aminoethanol and then with periodate.
The adipocyte Goα-immunoreactive polypeptide is different from the α subunit of the brain Go protein
Authors: Rouot B , Carrette J , Lafontan M , Lan Tran P , Fehrentz J A , Bockaert J , Toutant M
Published: August 2015

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.
Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins
Authors: Antony A C , Kane M A , Krishnan S R , Kincade R S , Verma R S
Published: August 2015

Membrane-associated folate (pteroylglutamate, PteGlu)-binding proteins (FBPs) play an important role as PteGlu-transport proteins in malignant and normal human cells. Since high extracellular folate (PteGlu) concentrations (EFC) profoundly influenced uptake and toxicity of the anti-PteGlu methotrexate in malignant KB cells, we studied human cells to determine additional mechanisms for PteGlu uptake when the EFC was varied. At low EFC (less than 10 nM), the predominant mechanism for folate uptake in mature erythrocytes was through binding to externally oriented FBPs which were quantitatively insignificant (4-6 orders of magnitude lower) and of no apparent physiological relevance when compared with KB cells. However, the predominant mechanism of PteGlu accumulation at high EFC [10-250 nM] in intact erythrocytes and sealed right-side-out (RSO) ghosts was not FBP-mediated and non-specific. This conclusion was based on the findings that radiolabelled PteGlu uptake: (i) continued even in the presence of a 1000-fold excess of unlabelled PteGlu and was linear and not saturable up to 250 nM; (ii) was two-fold higher at pH 4.5 than 7.5; (iii) was less than 2-fold increased at 37 degrees C compared with 4 degrees C; and (iv) was unaffected after trypsin-mediated proteolysis of greater than 75% FBPs. The [3H]PteGlu and 125I-PteGlu (histamine derivative) accumulated intracellularly through the non-specific PteGlu-uptake mechanism was unaltered biochemically and in a soluble compartment. Raising the EFC 500-fold higher than controls during erythropoiesis in vitro resulted in reversal of the expected anti-(placental folate-receptor)-antiserum-induced megaloblastic changes in orthochromatic normoblasts derived from burst-forming unit-erythroid colonies. Furthermore, at EFC greater than 0.1 microM, KB-cell accumulation of [3H]PteGlu was also predominantly through a mechanism that did not involve specific FBPs. Thus, at high EFC, a major component of PteGlu transport in human cells is not mediated through FBPs and is likely to be a passive diffusion process.
Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit
Authors: Weyler W
Published: August 2015

I present the first clear evidence that the protein: FAD ratio in human monoamine oxidase A and bovine monoamine oxidase B has an upper limit of 65 kDa and 57 kDa per FAD, respectively. To now it had been assumed that the protein: FAD ratio was 100-120 kDa to 1 FAD and that there was one FAD per two subunits which were assumed to be of the same size. For the present work the purity of monoamine oxidase A and monoamine oxidase B was improved over that previously achieved. Protein was determined by quantitative amino acid analysis and FAD content was measured by spectrophotometric titration of SDS-denatured enzyme with NaS2O4 standardized against riboflavin. The cause of the previous misassignment of the protein: FAD ratio was judged as having been due to the use of impure enzyme preparations. Knowledge of the correct protein: FAD ratio is important in devising cloning strategies for this enzyme, in understanding its structure, function, mechanism, and in the studies of its biosynthesis.
The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1
Authors: Nunn D N , Day D , Anthony C
Published: August 2015

The nucleotide and deduced amino acid sequence of a novel small (beta) subunit of methanol dehydrogenase of Methylobacterium extorquens AM1 (previously Pseudomonas AM1) has been determined. Work with the whole protein has shown that is has an alpha 2 beta 2 configuration.
ω–3 fatty acids increase the arachidonic acid content of liver cholesterol ester and plasma triacylglycerol fractions in the rat
Authors: Garg M L , Wierzbicki A A , Thomson A B R , Clandinin M T
Published: August 2015

Recent studies have demonstrated that dietary fish oils rich in eicosapentaenoic acid (C20:5,omega 3) lower the content of arachidonic acid and its metabolites in plasma and tissue phospholipids. The present study examined the fatty acid composition of cholesterol ester and triacylglycerol fractions from plasma and livers of rats fed diets enriched with saturated fatty acids (beef tallow), alpha-linolenic acid (linseed oil) or eicosapentaenoic acid (fish oil). Feeding diets containing linseed oil or fish oil for 28 days increased arachidonic acid (C20:4,omega 6) levels in the cholesterol ester fraction of liver and in the triacylglycerol fraction of the plasma lipids. Plasma cholesterol esters were depleted of C20:4,omega 6 after feeding of the diet containing either linseed oil or fish oil. The changes in C20:4,omega 6 content cannot be explained by alterations in cholesterol ester or triacylglycerol pools of plasma and liver. These results suggest that the decrease in phospholipid C20:4,omega 6 content generally observed after fish oil consumption may be partly due to a shift of C20:4,omega 6 from phospholipid to the triacylglycerol and/or cholesterol ester pools in the same tissue. Triacylglycerols and cholesterol esters may therefore play a buffering role in the homeostatic maintenance of tissue phospholipid levels of arachidonic acid.
Carbachol and histamine stimulation of guanine-nucleotide-dependent phosphoinositide hydrolysis in rat brain cortical membranes
Authors: Claro E , Garcia A , Picatoste F
Published: August 2015

Guanine nucleotides have been shown to stimulate phosphoinositide breakdown in brain membranes, but no potentiation of such an effect by agonist was demonstrated. We have studied the effect of carbachol and histamine on guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulation of inositol phosphates formation in [3H]inositol-labelled rat brain cortical membranes. In this preparation, GTP[S] enhancement of phosphoinositide hydrolysis required the presence of MgATP and low Ca2+ concentration (100 nM). Carbachol potentiation of the GTP[S] effect was only observed when 1 mM-deoxycholate was also added. Under these conditions, stimulated production of [3H]inositol phosphates was linear for at least 15 min, and [3H]inositol bisphosphate [(3H]IP2) accounted for approx. 80%, whereas the amount of [3H]inositol trisphosphate [(3H]IP3) was very low. Stimulation by GTP[S] was concentration-dependent (half-maximal effect at 0.86 microM), and its maximal effect (815% over basal) was increased by 1 mM-carbachol (1.9-fold) and -histamine (1.7-fold). Both agonists decreased the slope index of the GTP[S] concentration/effect curve to values lower than unity, suggesting the appearance of some heterogeneity in the population of guanine-nucleotide-binding proteins (G-proteins) involved. The carbachol and histamine effects were also concentration-dependent, and were inhibited by atropine and mepyramine respectively. Fluoroaluminate stimulated phosphoinositide hydrolysis to a higher extent than GTP[S] plus carbachol, and these stimulations were not additive, indicating that the same polyphosphoinositide phospholipase C-coupled G-protein mediates both effects.
Interferon alters the pattern of secreted proteins from Ehrlich ascites-tumour cells
Authors: Tominaga S , Tominaga K
Published: August 2015

Treatment of Ehrlich ascites-tumour (EAT) cells with interferon (IFN) abolished their ability to secrete a 32 kDa protein that was secreted by growing EAT cells. These IFN-treated cells secreted two proteins (molecular masses 100 and 89 kDa as estimated by SDS/polyacrylamide-gel electrophoresis) that were not detected in two-dimensional gel electrophoresis of the culture fluid of untreated EAT cells. The sequence of 20 amino acids from the N-terminal end of the 32 kDa protein was very similar to portions of sequences of mouse proviral gag proteins.
Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5′-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells
Authors: Holm I , Persson L , Pegg A E , Heby O
Published: August 2015

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5′-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.
Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases
Authors: Alin P , Jensson H , Cederlund E , Jörnvall H , Mannervik B
Published: August 2015

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.
Complete localization of the intrachain disulphide bonds and the N-glycosylation points in the α-subunit of human platelet glycoprotein IIb
Authors: Calvete J J , Henschen A , González-Rodríguez J
Published: August 2015

Glycoprotein IIb (GPIIb), one of the two molecular components of the inducible receptor for fibrinogen on the platelet surface, is formed from two subunits, GPIIb alpha (114 kDa) and GPIIb beta (22.5 kDa), joined by a single disulphide bond. CNBr cleavage of GPIIb, together with tryptic or endoproteinase Lys-C digestion of some of the isolated CNBr peptides, followed by amino acid and N-terminal sequence analysis of the isolated fragments, allowed us to locate unambiguously all the unknown disulphide bonds and the N-glycosylation points in platelet GPIIb. It could be established that each cysteine residue in GPIIb, beginning at alpha-Cys-56, is disulphide-bonded to its nearest neighbour in the amino acid sequence. Given the extensive structural similarity among the two-chain alpha-subunits of Arg-Gly-Asp adhesion receptors and the conservative positions of cysteine residues in their amino acid sequences, the intrachain and interchain disulphide-bond pattern found here in GPIIb will most probably be conserved in all two-chain alpha-subunits of these receptors. The N-linked glycosylation points found here in platelet GPIIb are the same as the five N-glycosylated asparagine residues suggested after cDNA sequencing of human erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482]. Some of the general features of the structure of GPIIb, such as the existence of distinct domains in the alpha- and beta-subunits, as well as the identification of well-defined points in its external topography, are discussed.
High activity of cytochrome P-450-linked aminopyrine N-demethylase in mouse brain microsomes, and associated sex-related difference
Authors: Ravindranath V , Ananda Theertha Varada H K
Published: August 2015

The presence of cytochrome P-450 and associated mono-oxygenase activities was examined in brain microsomes from male and female mice. Although the cytochrome P-450 level in male mouse brain was very low as compared with mouse liver, the aminopyrine N-demethylase and morphine N-demethylase specific activities in male mouse brain were much higher than those observed in mouse liver. Ethoxycoumarin O-de-ethylase and aniline hydroxylase activities were, however, not detected in mouse brain. Sex-related differences were observed in both the cytochrome P-450 levels and aminopyrine N-demethylase activity in mouse brain, the levels of both being higher in male mouse brain as compared with female mouse brain. Aminopyrine N-demethylase activity in mouse brain microsomes was dependent on the presence of oxygen and NADPH and could be inhibited by piperonyl butoxide, N-octyl imidazole and carbon monoxide. Antiserum raised to the phenobarbital-inducible form of rat liver cytochrome P-450 [P-450(b+e)] inhibited mouse brain aminopyrine N-demethylase activity by around 80+ mouse brain microsomal protein exhibited cross-reactivity against this antiserum when examined by Ouchterlony double diffusion and immunoblotting. The present results indicate the presence of a phenobarbital-inducible form of cytochrome P-450 (or a form of cytochrome P-450 that is similar immunologically) in mouse brain microsomes, which is associated with a sex-related difference.
Further kinetic and molecular characterization of an extremely heat-stable carboxylesterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: Sobek H , Görisch H
Published: August 2015

The carboxylesterase (serine esterase, EC 3.1.1.1) from Sulfolobus acidocaldarius was purified 940-fold to homogeneity by an improved purification procedure with a yield of 57%. In the presence of alcohols the enzyme catalyses the transfer of the substrate acyl group to alcohols in parallel to hydrolysis. The results show the existence of an alcohol-binding site and a competitive partitioning of the acyl-enzyme intermediate between water and alcohols. Aniline acts also as a nucleophilic acceptor for the acyl group. On the basis of titration with diethyl p-nitrophenyl phosphate, a number of four active centres is determined for the tetrameric carboxylesterase. The sequence of 20 amino acid residues at the esterase N-terminus and the amino acid composition are reported.
Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein β) gene. A further study of alterations in the glycophorin C gene
Authors: High S , Tanner M J A , Macdonald E B , Anstee D J
Published: August 2015

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5′ and 3′ flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.
The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents
Authors: Angliker H , Wikstrom P , Kirschke H , Shaw E
Published: August 2015

An attempt has been made to extend to the cysteinyl exopeptidases cathepsins H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsins B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidyl aminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 microM-H2N-Phe-CH2F (the ‘H2N’ indicates a free unblocked amino group) (k2 = 1878 M-1.s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivatives H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10(-7) M. Higher concentrations inactivated cathepsins H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C.
Inhibition of cholesterol 7α-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC
Authors: Eldredge E R , Jackson B , Suckling K E , Wolf C R
Published: August 2015

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.
Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinyl-CoA content in liver
Authors: Quant P A , Tubbs P K , Brand M D
Published: August 2015

1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase.
Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein α-fragment
Authors: Stillman M J , Zelazowski A J
Published: August 2015

The yield of the alpha-fragment of rabbit liver metallothionein 2 was used to test the domain-specificity and mobility of Cd2+ and Zn2+ when bound to metallothionein. Increasing molar ratios of Cd2+ were added to either Zn7-metallothionein or the metal-ion-free apo-metallothionein. The enzyme subtilisin was used to digest those parts of the peptide chain that were not bound to Cd2+. Analysis of the digestion products was carried out by separation by polyacrylamide-gel electrophoresis. The chelation agent EDTA was used as a competitive chelator. It was found that the presence of excess EDTA greatly enhances the formation of the Cd4-metallothionein alpha-fragment, and catalyses the complete digestion of all other the metal-ion-containing peptides, so that even Cd7-metallothionein, formed when 7 molar equivalents of Cd2+ are added to Zn7-metallothionein, is digested to the alpha-fragment. These results suggest that the Cd2+ bound in the beta-sites is very labile, much more labile than the kinetics of the off-reaction would suggest. The observation of significant amounts of alpha-fragment on the gels, even when the stoichiometry of the metal ions initially present in the protein should not have resulted in much concentration of Cd4-alpha-fragment clusters, indicates that as the digestion proceeds the metal ions move to sites that form complete clusters and therefore selectively protect that part of the peptide chain from digestion. We also find that rabbit Cd4-metallothionein 2 alpha-fragment stains near to the top of the gel, in complete contrast with the location of rat Cd4-metallothionein 2 alpha-fragment. This difference in the mobilities suggests that the alpha-fragment prepared from rabbit metallothionein 2 is much less negatively charged than the analogous protein fragment prepared from rat liver metallothionein 2.
Physicochemical studies of binding of 4-methylumbelliferyl β-d-galactopyranoside to cold agglutinin
Authors: Mitra D , Sarkar M
Published: August 2015

The fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmbGalp) was quenched in the presence of cold agglutinin, showing that there was binding between MeUmbGalp and cold agglutinin. That binding was saccharide-specific. By using this quenching phenomenon, the association constants (Ka) of the binding of cold agglutinin at different temperatures (10 degrees C and 15 degrees C) to MeUmbGalp and also the number of binding sites were calculated. The Ka values were found to be 2.63 x 10(3) M-1 at 10 degrees C and 1.58 x 10(3) M-1 at 15 degrees C. Though there is a change in Ka values, the number of binding sites was calculated to be six at both temperatures (10 degrees C and 15 degrees C). From the Ka values the thermodynamic parameters (free energy, enthalpy and entropy) of the binding were derived, and analysis of the data indicated that the binding is spontaneous, exothermic and hydrophobic in nature.
Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp
Authors: Saravani G A , Cowan D A , Daniel R M , Morgan H W
Published: August 2015

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.
Glucagon, cyclic AMP and adrenaline stimulate the degradation of low-density lipoprotein by cultured rat hepatocytes
Authors: Brown N F , Salter A M , Fears R , Brindley D N
Published: August 2015

Rat hepatocytes were preincubated for 16 h with hormones or drugs and then for a further 8 h with 125I-human low-density lipoprotein (LDL). Glucagon (via cyclic AMP) and adrenaline (via cyclic AMP and alpha-effects) increased the binding of 125I-LDL to the LDL receptor, and the degradation of LDL to [125I]iodotyrosine. The effects on degradation were antagonized by dexamethasone, and the action of cyclic AMP on binding and degradation was inhibited by actinomycin D. The results are discussed in relation to the control of lipoprotein metabolism in diabetes.
Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells
Authors: Hanekom C , Nel A , Gittinger C , Rheeder A , Landreth G
Published: August 2015

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.
Presence of haemin-controlled eIF-2α kinases in both undifferentiated and differentiating mouse erythroleukaemia cells
Authors: Sarre T F
Published: August 2015

In rabbit reticulocytes, globin synthesis is regulated by a haemin-controlled translational inhibitor (HCI) which acts by phosphorylating the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). With purified eIF-2 as substrate, haemin-controlled eIF-2 alpha kinases could be partially purified from cultured mouse erythroleukaemia cells (MEL cells), which can be induced in vivo to erythroid differentiation. The eIF-2 alpha kinases from both uninduced and induced MEL cells are clearly distinct from the double-stranded-RNA-activated eIF-2 alpha kinase described for many mammalian cell types. A rough quantitative estimation indicates that, on a per-cell basis, induced MEL cells contain the same amount of haemin-controlled eIF-2 alpha kinase activity as rabbit reticulocytes, whereas uninduced MEL cells contain about one-tenth as much. As to their chromatographic behavior on CM-Sephadex and DEAE-cellulose and their sensitivity towards physiological concentrations of haemin (5-10 microM), the eIF-2 alpha kinases from MEL cells are indistinguishable from HCI. They differ from HCI with respect to their response towards activating stimuli such as prolonged incubation at 37 degrees C or brief exposure to the thiol reagent N-ethylmaleimide.
A kinetic study of the suicide inactivation of an enzyme measured through coupling reactions. Application to the suicide inactivation of tyrosinase
Authors: Escribano J , Tudela J , Garcia-Carmona F , Garcia-Canovas F
Published: August 2015

A systematic procedure for the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or in the absence of an auxiliary substrate, when the enzyme activity is measured through coupling reactions, enzymically catalysed or not, was developed and analysed by using the transient-phase approach. The methodology is established to determine the parameters and kinetic constants corresponding to the enzyme suicide inactivation and the coupling reactions. This approach is illustrated by a study of the suicide inactivation of tyrosinase by catechol in the presence of L-proline. Treatment of the experimental data was carried out by non-linear regression.
Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase
Authors: Anderson N G , Kilgour E , Houslay M D
Published: August 2015

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous ‘dense-vesicle’ cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver ‘dense-vesicle’ cyclic AMP phosphodiesterase.
Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors
Authors: Doherty F J , Osborn N U , Wassell J A , Heggie P E , Laszlo L , Mayer R J
Published: August 2015

Mouse fibroblasts (3T3-L1 cells) accumulate detergent- and salt-insoluble aggregates of proteins conjugated to ubiquitin when incubated in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. These ubiquitin-protein conjugates co-fractionate with lysosomes on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. Both E-64 and ammonium chloride increase the intracellular concentration of free ubiquitin, but only E-64 leads to the formation of insoluble lysosomal ubiquitin-protein conjugates. The results are discussed in relation to the possible intracellular roles of ubiquitin conjugation.
Phospholipase C-mediated hydrolysis of phosphatidylcholine is activated by muscarinic agonists
Authors: Diaz-Meco M T , Larrodera P , Lopez-Barahona M , Cornet M E , Barreno P G , Moscat J
Published: August 2015

The phospholipase C-catalysed breakdown of inositol-containing phospholipids is an important source of diacylglycerol in cells stimulated by several agonists. However, recent experimental evidence suggests that major phospholipids such as phosphatidylcholine may also be substrates of the phosphodiesteratic hydrolysis activated by hormones, growth factors and oncogene products. We show here that stimulation of muscarinic agonists activates the release of phosphocholine, which, along with diacylglycerol, is a metabolic product of phospholipase C-mediated hydrolysis of phosphatidylcholine. Fluoroaluminates mimic this muscarinic effect, strongly suggesting that carbachol-activated release of phosphocholine may be mediated by a guanine-nucleotide-binding protein. Evidence for this was obtained from experiments using permeabilized cells in which non-hydrolysable analogues of GTP activated phosphocholine release synergistically with carbachol.
Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments
Authors: Leyte A , Mertens K , Distel B , Evers R F , De Keyzer-Nellen M J M , Groenen-Van Dooren M M C L , De Bruin J , Pannekoek H , Van Mourik J A , Verbeet M P
Published: August 2015

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.
Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism
Authors: Dubaniewicz A , Kaciuba-Usciłko H , Nazar K , Budohoski L
Published: August 2015

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.