bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking
Authors: Patel S D , Ragan C I
Published: August 2015

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.
Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking
Authors: Patel S D , Cleeter M W J , Ragan C I
Published: August 2015

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.
Rapid kinetic studies of the reduction of cellobiose oxidase from the white-rot fungus Sporotrichum pulverulentum by cellobiose
Authors: Jones G D , Wilson M T
Published: August 2015

The reactions between cellobiose and cellobiose oxidase were investigated by stopped-flow spectrophotometry. Under anaerobic conditions rapid reduction of the associated flavin is followed by slower reduction of cytochrome b. The kinetic difference spectra are reported. The rate of flavin reduction depends on the cellobiose concentration (with an apparent second-order rate constant of approx. 10(5) M-1.s-1) but reaches a rate limit of approx. 20 s-1. In contrast, the rate of cytochrome b reduction decreases at high cellobiose concentrations. Kinetic titrations of the flavin and cytochrome b moieties yield the stoichiometries of the separate reactions, i.e. the number of moles of cellobiose needed to fully reduce 1 mole of each redox component. The rate constant for cytochrome b reduction, unlike that for flavin reduction, increased with enzyme concentration, prompting the conclusion that any given cytochrome b centre is reduced preferentially by flavin groups in different molecules rather than by its partner flavin within the same monomer. These data are discussed in the context of a scheme that rationalizes them and accounts for the overall stoichiometry in which three two-electron donors (cellobiose molecules) reduce two three-electron acceptors (the flavin-cytochrome b of cellobiose oxidase).
Insulin-receptor phosphotyrosyl-protein phosphatases
Authors: King M J , Sale G J
Published: August 2015

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) phosphatases.
Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum
Authors: Wuytack F , Kanmura Y , Eggermont J A , Raeymaekers L , Verbist J , Hartweg D , Gietzen K , Casteels R
Published: August 2015

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.
Structural similarity of chymopapain forms as indicated by circular dichroism
Authors: Solis-Mendiola S , Zubillaga-Luna R , Rojo-Dominguez A , Hernandez-Arana A
Published: August 2015

Four chymopapain forms were isolated by high-resolution liquid chromatography on a cation-exchange column. The three major forms possess nearly identical secondary and tertiary structures, as judged from their c.d. spectra; these components showed similar proteolytic activity and Mr values close to that of papain. The fourth isolated component seems to be a mixture of modified proteins.
Purification of a growth factor related to platelet-derived growth factor and a type β transforming growth factor secreted by mouse neuroblastoma cells. A general strategy for the purification of basic polypeptide growth factors
Authors: Van den Eijnden-Van Raaij A J M , Koornneef I , Van Oostwaard T M J , Feyen A , Kruijer W , De Laat S W , Van Zoelen E J J
Published: August 2015

A general strategy was developed for the purification of basic polypeptide growth factors. This method is a combination of gel filtration, weak-cation-exchange h.p.l.c. and reverse-phase h.p.l.c., separating the proteins according to size, charge and hydrophobicity respectively. All steps are carried out at low pH with exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by freeze-drying facilitate the detection of the growth factors by biological assays. By using this method, homogeneous preparations of two basic growth factors were purified in high yield from mouse-neuroblastoma-Neuro-2A-cell-conditioned medium. It is shown that these purified factors are biochemically and immunologically related to platelet-derived growth factor and type beta transforming growth factor from human platelets.
Caprine acrosin. Purification, characterization and proteolysis of the porcine zona pellucida
Authors: Hardy D M , Schoots A F M , Hedrick J L
Published: August 2015

Acrosin purified from an acidic extract of ejaculated goat spermatozoa migrated as a single 42,000-Mr band in SDS/polyacrylamide-gel electrophoresis. Reduction and alkylation of caprine acrosin produced two polypeptides, one of Mr 40,000 (heavy chain) and the other of Mr 3700 (light chain). The light chain purified by reversed-phase h.p.l.c. was a glycosylated octadecapeptide with an amino acid sequence similar to that of the N-terminal 18 residues of porcine acrosin light chain (78% positional identity). The sequence of the N-terminal 37 amino acids of purified caprine acrosin heavy chain is similar to that of porcine acrosin heavy chain (70% positional identity through 37 residues). Studies with synthetic substrates and synthetic and natural proteinase inhibitors confirmed both the specificity of the purified proteinase for Arg-Xaa and Lys-Xaa bonds and a serine-proteinase mechanism. Purified caprine acrosin hydrolysed the 90 kDa and 65 kDa components, but did not hydrolyse the 55 kDa component of the porcine zona pellucida. The action of the enzyme on the porcine zona pellucida was indistinguishable from that previously reported for porcine acrosin.
The action of cytoplasmic aldehyde dehydrogenase on methyl p-nitrophenyl carbonate and p-nitrophenyl dimethylcarbamate
Authors: Kitson T M
Published: August 2015

Cytoplasmic aldehyde dehydrogenase catalyses the hydrolysis of methyl p-nitrophenyl (PNP) carbonate at an appreciable rate that is markedly stimualted by NAD+ or NADH. The nuleotides accelerate the rate-limiting hydrolysis of the acyl-enzyme intermediate while slowing the observed burst of p-nitrophenoxide production. With PNP dimethylcarbamate the enzyme catalyses the slow release of approx. 1 molecule of p-nitrophenoxide per tetrameric enzyme molecule; during the reaction the enzyme becomes effectively inactivated, as the rate of hydrolysis of the acyl-enzyme is virtually zero. The presence of NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol or disulfiram slows the reaction of enzyme with PNP dimethylcarbamate. The reaction appears to be dependent on a group of pKa 7.6, possibly a cysteine residue. The effect of PNP dimethylcarbamate on the dehydrogenase activity of the enzyme is consistent with there being a single type of active site for the enzyme's dehydrogenase and esterase activities. Steric and electronic factors that govern reaction of the enzyme with PNP substrates are discussed.
Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases
Authors: Siess W , Lapetina E G
Published: August 2015

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.
Effect of cis-unsaturated fatty acids on aortic protein kinase C activity
Authors: Dell K R , Severson D L
Published: August 2015

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.
Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue
Authors: Frick G P , Goodman H M
Published: August 2015

The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.
‘Star’ activity and complete loss of specificity of CeqI endonuclease
Authors: Izsvák Z , Duda E
Published: August 2015

Restriction endonuclease CeqI, an isoschizomer of EcoRV, exhibits ‘star’ activity, a relaxation of specificity in the presence of Mn2+, dimethyl sulphoxide or glycerol. The enzyme cleaves a set of sequences that differ from the canonical GATATC by only one nucleotide in positions 2, 3, 4 or 5. Two of these sequences are not cleaved if modified by dam methylase. A further loss of specificity can be observed in circumstances less favourable for the enzyme, namely low-ionic-strength buffers of pH values below 6.0 or above 9.4. This activity seems to cleave DNA at any sequence, producing a smear instead of well-defined bands. Partial renaturation of the denatured enzyme gives rise to a similar non-specific nuclease activity.
Effect of noradrenaline on triacylglycerol synthesis in rat brown adipocytes
Authors: Baht H S , Saggerson E D
Published: August 2015

Incubation of rat brown adipocytes with noradrenaline in the presence of insulin and palmitate caused a decrease in the rate of triacylglycerol synthesis as measured by [U-14C]glucose incorporation into acylglycerol glycerol. Concomitantly, the ratio of [1-14C]palmitate oxidized to CO2 to that esterified was increased. This alteration in the rate of triacylglycerol synthesis by noradrenaline was not observed when fatty acid oxidation was inhibited by etomoxir. Noradrenaline did not cause any acute inactivation of enzymes of the triacylglycerol-synthesis pathway. It is suggested that the decrease in triacylglycerol synthesis seen with noradrenaline is secondary to activation of fatty acid oxidation.
Capacitative calcium entry in parotid acinar cells
Authors: Takemura H , Putney J W
Published: August 2015

The intracellular Ca2+ indicator, fura-2, was used to monitor changes in cytosolic [Ca2+] in parotid acinar cells. When parotid cells were incubated in a medium containing low [Ca2+], and [Ca2+] was restored to the physiological range, there was a small increase in cytosolic [Ca2+]. If, however, the cells were first activated by a muscarinic agonist, and receptor activation was terminated before the addition of Ca2+ by the addition of a pharmacological excess of the muscarinic-receptor antagonist atropine, the initial increase in cytosolic [Ca2+] was faster and transiently larger than in the control cells which had not been previously stimulated. This suggested that a stimulation of Ca2+ entry occurred owing to the prior emptying of the agonist-regulated intracellular Ca2+ pool. This extra Ca2+ influx seen in pool-depleted cells persisted even when the interval between the addition of atropine and Ca2+ was increased from 1 to 20 min. Also, when the pool was allowed to refill by adding atropine in the presence of extracellular Ca2+, and Ca2+ was then sequentially removed and restored, the rise in cytosolic [Ca2+] after the addition of extracellular Ca2+ was not rapid, and resembled the increase seen in unstimulated cells. These results indicate that, when the agonist-sensitive Ca2+ pool is emptied by an agonist, Ca2+ influx across the plasma membrane is increased. This influx of Ca2+ occurs independently of the concentrations of inositol phosphates and probably of any second messengers linked directly to receptor activation. It appears rather to be a consequence of the empty state of the Ca2+ pool. Further, we suggest that, whenever the agonist-sensitive Ca2+ pool is emptied by agonist activation, the plasma-membrane permeability to Ca2+ will be increased, and this may account, at least in part, for the phenomenon of receptor-activated Ca2+ entry.
Inhibition of mitochondrial-matrix inorganic pyrophosphatase by physiological [Ca2+], and its role in the hormonal regulation of mitochondrial matrix volume
Authors: Davidson A M , Halestrap A P
Published: August 2015

1. The pyrophosphatase activity in cytosolic and mitochondrial fractions of rat liver was 1.7 and 0.26 units/mg of protein respectively when assayed at 37 degrees C in the presence of physiological [Mg2+] (0.3 mM). 2. Approx. 80% of the mitochondrial pyrophosphatase was inaccessible to extramitochondrial PPi, of which 40% represented soluble matrix enzyme (0.38 unit/mg of matrix protein). 3. Ca2+ inhibited the soluble matrix enzyme; the effective K0.5 for inhibition increased as [Mg2+], an essential cofactor of the enzyme, increased. Measured values were 0.39, 1.15, 3.7, 8.3 and 12.5 microM at 0.04 mM-, 0.1 mM-, 0.3 mM-, 0.6 mM- and 1 mM-Mg2+ respectively. 4. The data were analysed by a kinetic model similar to that for yeast pyrophosphatase, which assumes the substrate to be MgPPi (Km 5 microM) with Mg2+ also activating at an additional site (K0.5 23 microM). Ca2+ inhibits through the formation of CaPPi, a strong competitive inhibitor (Ki 0.067 microM). 5. Heart mitochondria also contain a soluble matrix pyrophosphatase of similar activity to that of liver mitochondria and with the same sensitivity to [Ca2+]. 6. The data provide an explanation for the increase in mitochondrial PPi, mediated by Ca2+, which is responsible for the increase in matrix volume induced by gluconeogenic hormones [Davidson & Halestrap (1988) Biochem. J. 254, 379-384].
Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase
Authors: Heeley D H , Smillie L B , Lohmeier-Vogel E M
Published: August 2015

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.
Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle
Authors: Salviati G , Betto R , Ceoldo S , Biasia E , Bonilla E , Miranda A F , Dimauro S
Published: August 2015

We studied the subcellular localization of dystrophin in rabbit skeletal muscle. In Western-blot analysis of membrane preparations, dystrophin was associated with the sarcolemmal fraction, as indicated by cholesterol content and co-purification with ouabain-binding activity and beta-adrenergic receptor. Dystrophin was also found with junctional T-tubules, but not with ‘free’ T-tubules, longitudinal portions or terminal cisternae of the sarcoplasmic reticulum. Dystrophin was not solubilized by high salt solutions, but it was solubilized by low concentrations of detergents (Triton X-100 and deoxycholate), suggesting that it is a peripheral membrane protein.
Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ
Authors: Nguyen Q , Murphy G , Roughley P J , Mort J S
Published: August 2015

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.
Comparison of the inhibition of insulin release by activation of adenosine and α2-adrenergic receptors in rat β-cells
Authors: Bertrand G , Nenquin M , Henquin J C
Published: August 2015

Rat islets were used to compare the mechanisms whereby adenosine and adrenaline inhibit insulin release. Adenosine (1 microM-2.5 mM) and its analogue N6(-)-phenylisopropyladenosine (L-PIA) (1 nM-10 microM) caused a concentration-dependent but incomplete (45-60%) inhibition of glucose-stimulated release. L-PIA was more potent than D-PIA [the N6(+) analogue], but much less than adrenaline, which caused nearly complete inhibition (85% at 0.1 microM). 8-Phenyltheophylline prevented the inhibitory effect of L-PIA and 50 microM-adenosine, but not that of 500 microM-adenosine or of adrenaline. In contrast, yohimbine selectively prevented the inhibition by adrenaline. Adenosine and L-PIA thus appear to exert their effects by activating membrane A1 receptors, whereas adrenaline acts on alpha 2-adrenergic receptors. Adenosine, L-PIA and adrenaline slightly inhibited 45Ca2+ efflux, 86Rb+ efflux and 45Ca2+ influx in glucose-stimulated islets. The inhibition of insulin release by adenosine or L-PIA was totally prevented by dibutyryl cyclic AMP, but was only attenuated when adenylate cyclase was activated by forskolin or when protein kinase C was stimulated by a phorbol ester. Adrenaline, on the other hand, inhibited release under these conditions. It is concluded that inhibition of adenylate cyclase, rather than direct changes in membrane K+ and Ca2+ permeabilities, underlies the inhibition of insulin release induced by activation of A1-receptors. The more complete inhibition mediated by alpha 2-adrenergic receptors appears to result from a second mechanism not triggered by adenosine.
Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets
Authors: Colard O , Breton M , Pepin D , Chevy F , Bereziat G , Polonovski J
Published: August 2015

The origin of the arachidonate released from platelets on stimulation with thrombin was investigated by comparing the specific activities of released arachidonate and of arachidonoyl-containing phospholipids using rat platelets prelabelled with arachidonate. Quantification of the released arachidonate was determined in the presence of BW 755 C, a dual cyclo-oxygenase/lipoxygenase inhibitor, which was found not to modify the arachidonate mobilization between the platelet phospholipids. The phospholipid molecular species were analysed by h.p.l.c. of diradylglycerol benzoate derivatives of diacyl, alkylacyl and alkenylacyl classes. The labelled/unlabelled arachidonate ratio varied greatly in the phospholipids depending on whether an ether or acyl bond was present in sn-1 position of the glycerol, on the length and degree of unsaturation of this fatty chain and on the polar head group. Between 15 s and 5 min of stimulation by thrombin, the released arachidonate kept a constant specific activity which was considerably lower than the specific activity of diacyl-GPC. The specific activity of the released arachidonate was intermediate between the specific activities of the 16:0-20:4 and 18:0-20:4 species of diacyl-GPI and diacyl-GPE, and corresponded to the mean specific activity of alkylacyl-GPC. The data indicate that the released arachidonate cannot come directly from diacyl-GPC, and that two phospholipids in particular can act as direct precursors of the released arachidonate. These are (1) the alkylacyl-GPC and (2) the diacyl-GPE whose hydrolysis would induce an arachidonate transfer from diacyl-GPC.
Lack of V1 vasopressin receptors in rabbit hepatocytes
Authors: Vandekerckhove A , Miot F , Keppens S , De Wulf H
Published: August 2015

Vasopressin does not induce glycogenolysis in rabbit hepatocytes; glucagon, angiotensin, phenylephrine and ATP are as potent as with rat hepatocytes, whereas isoprenaline is nearly 10000 times more potent in the rabbit. Binding studies of [3H]vasopressin reveal the complete absence of specific vasopressin receptors on rabbit liver plasma membranes. We verified that vasopressin acts as an antidiuretic and vasopressor agent in the rabbit. We conclude that there is a selective lack of V1 vasopressin receptors in rabbit liver.
Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver
Authors: Vanstapel F , Hammaker L , Pua K , Blanckaert N
Published: August 2015

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.
A key functional role for the insulin-like growth factor 1 N-terminal pentapeptide
Authors: Bagley C J , May B L , Szabo L , McNamara P J , Ross M , Francis G L , Ballard F J , Wallace J C
Published: August 2015

In order to elucidate the role of the N-terminus of insulin-like growth factor 1 (IGF-1) with respect to its biological properties, we chemically synthesized analogues of IGF-1 truncated by one to five amino acid residues from the N-terminus. In a bioassay that measured the stimulation of protein synthesis in rat L6 myoblasts, the concentrations required to produce a half-maximal response were: IGF-1, 13 ng/ml; des-(1)-IGF-1, 10 ng/ml; des-(1-2)-IGF-1, 13 ng/ml; des-(1-3)-IGF-1, 1.5 ng/ml; des-(1-4)-IGF-1, 5.1 ng/ml; des-(1-5)-IGF-1, 1200 ng/ml. When tested for their abilities to compete with 125I-IGF-1 binding to L6 myoblasts at 3 degrees C, the concentrations required for 50% competition were: IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1, 20 ng/ml; des-(1-3)-IGF-1, 14 ng/ml; des-(1-4)-IGF-1, 40 ng/ml; des-(1-5)-IGF-1, greater than 1000 ng/ml. Receptor-binding experiments at 25 degrees C, however, gave results suggesting that the myoblasts were secreting a binding protein selective for the three longest peptides. This interpretation was confirmed by binding studies with medium conditioned by the L6 myoblasts as well as binding protein purified from MDBK-cell-conditioned medium. In both cases IGF-1, des-(1)-IGF-1 and des-(1-2)-IGF-1 competed for tracer IGF-1 binding at least 60-fold better than did the three shorter peptides. The results obtained account for the increased potency of des-(1-3)-IGF-1 and des-(1-4)-IGF-1, since their activities are not attenuated by the binding protein, and the relatively lower potency of des-(1-4)-IGF-1 is a consequence of this peptide binding less well to the L6-myoblast receptor.
Mechanism of hypoglycaemic action of methylenecyclopropylglycine
Authors: Melde K , Buettner H , Boschert W , Wolf H P O , Ghisla S
Published: August 2015

The effects of methylenecyclopropylglycine (MCPG), the lower homologue of hypoglycin A, on starved rats are described. Upon oral ingestion of MCPG (43 mg/kg), a 50% decrease in blood glucose compared with controls was observed after 4 h. The plasma concentrations of lactate and non-esterified fatty acids were substantially increased during this period. The activity of general acyl-CoA dehydrogenase from isolated rat liver mitochondria was not significantly changed. By contrast, the activity of 2-methyl-(branched-chain)-acyl-CoA dehydrogenase decreased by over 80%. The enzyme activity of enoyl-CoA hydratase (crotonase) from pig kidneys decreased by 80% on incubation with the hypothetically toxic metabolite of MCPG, methylenecyclopropylformyl-CoA. These results suggest that the inhibition spectrum of MCPG is quite different from that of hypoglycin A and that similar physiological effects might result from inhibition of different enzymes of beta-oxidation, e.g. hypoglycaemia and lacticacidemia. Accumulation of medium-chain acyl-CoA thioesters is probably at the origin of disturbances in pyruvate metabolism.
Identification of the ATP + Mg-dependent and polycation-stimulated protein phosphatases in the germinal vesicle of the Xenopus oocyte
Authors: Jessus C , Goris J , Staquet S , Cayla X , Ozon R , Merlevede W
Published: August 2015

Two protein phosphatase activities were characterized in the germinal vesicle of Xenopus laevis oocytes after manual dissection of the nucleus. One enzyme can be classified as an active form of the ATP + Mg-dependent (AMD) phosphatase, the other as a polycation-stimulated (PCS) phosphatase. The activity of the PCS phosphatase is localized exclusively in the soluble compartment of the nucleus (nucleoplasm). The catalytic subunit of the AMD phosphatase activity is associated either with the nuclear particulate fraction or with an inhibitory subunit in the nucleoplasm.
Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding
Authors: Wendland M , Hille A , Nagel G , Waheed A , von Figura K , Pohlmann R
Published: August 2015

The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.
Identification of cysteine residues in carbamoyl-phosphate synthase I with reactivity enhanced by N-acetyl-l-glutamate
Authors: Geschwill K , Lumper L
Published: August 2015

Carbamoyl-phosphate synthase I (pig liver) is modified at the cysteine residues 1327 and 1337 (numbered according to the rat sequence) in the presence of 5 mM-N-acetyl-L-glutamate with enhanced rate. ATP/Mg2+ (greater than or equal to 5 mM) protects against alkylation of these two cysteines and loss of activity. According to the results obtained by limited proteolysis of monobromobimane-modified carbamoyl-phosphate synthase I, the accessible cysteines 1327 and 1337 are located in the C-terminal 20 kDa domain D of the enzyme. N-Bromoacetyl-L-glutamate is an allosteric activator and inactivates carbamoyl-phosphate synthase in a slow reaction.
A carboxypeptidase Y pulse method to study the accessibility of the C-terminal end during the refolding of ribonuclease A
Authors: Teschner W , Rudolph R
Published: August 2015

Carboxypeptidase Y pulses, applied after various times of refolding, were employed to probe the accessibility of the C-terminus of RNAase A during the refolding process. The increase in resistance against proteolytic cleavage was measured by determination of the amount of liberated C-terminal amino acids and by activity assays. The results indicate that the C-terminus of RNAase becomes inaccessible early in the course of refolding, if folding is carried out at low temperatures under conditions that effectively stabilize the native state. At higher temperatures (25 degrees C) or under conditions of marginal stability, intermediates are not populated and protection against proteolytic cleavage is not detectable before the formation of the native state. The method described may be used to monitor the accessibility of the C-terminus of various proteins during refolding. However, intermediates on the folding pathway can only be observed if the native state is stable against carboxypeptidase attack.
The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region
Authors: Leyh-Bouille M , Van Beeumen J , Renier-Pirlot S , Joris B , Nguyen-Distèche M , Ghuysen J M
Published: August 2015

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of the conserved tetrad Ser-Xaa-Xaa-Lys occurs at position 35. There is no indication for the presence of a signal peptide or an N-terminal hydrophobic sequence, suggesting that the Streptomyces K15 enzyme is probably anchored to the membrane by a C-terminal peptide segment.
Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells
Authors: Squinto S P , Jungmann R A
Published: August 2015

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.
MK-801 is a potent nematocidal agent. Characterization of MK-801 binding sites in Caenorhabditis elegans
Authors: Schaeffer J M , Bergstrom A R , Turner M J
Published: August 2015

MK-801, an N-methyl-D-aspartate antagonist in mammalian brain tissue, is a potent nematocidal agent. Specific MK-801 binding sites have been identified and characterized in a membrane fraction prepared from the free-living nematode Caenorhabditis elegans. The high-affinity MK-801 binding site has an apparent dissociation constant, Kd, of 225 nM. Unlike the MK-801 binding site in mammalian tissues, the C. elegans binding site is not effected by glutamate or glycine, and polyamines are potent inhibitors of specific MK-801 binding.
Discovery of a protein phosphatase activity encoded in the genome of bacteriophage λ. Probable identity with open reading frame 221
Authors: Cohen P T W , Cohen P
Published: August 2015

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.
Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia
Authors: Edmonds C , Griffin G E , Johnstone A P
Published: August 2015

ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins. To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum. The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe. The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi). The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity. The modification is mono-ADP-ribosyl in nature. The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond. The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein. The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought.
Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells
Authors: Franklin T J , Hitchen M
Published: August 2015

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. 2,7,8-Trihydroxyanthraquinone (THA) is an effective inhibitor of prolyl 4-hydroxylase by virtue of its ability to compete with the co-substrate 2-oxoglutarate (Ki = 40.3 microM). Using a simple and reproducible assay for collagen hydroxylation, we show that THA inhibits the hydroxylation of collagen in embryonic-chick tendon cells in short-term culture, with an IC50 value (concn. giving 50% inhibition) of 32 microM. In comparison, the ethyl ester of 3,4-dihydroxybenzoic acid has an IC50 value of 0.1 mM against collagen hydroxylation by chick tendon cells, whereas its Ki versus isolated prolyl 4-hydroxylase is 49 microM.