bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase activity of the insulin receptor
Authors: Gammeltoft S , Van Obberghen E
Published: August 2015

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
GM1 gangliosidosis (type 1) in a cat
Authors: Barker C G , Blakemore W F , Dell A , Palmer A C , Tiller P R , Winchester B G
Published: August 2015

A kitten with clinical and morphological symptoms of a neurovisceral lysosomal-storage disease has been shown to have a marked deficiency of acidic beta-D-galactosidase in the brain, kidney and spleen. Chromatography on concanavalin A-Sepharose and inhibition studies with 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, a selective inhibitor of the neutral broad-specificity beta-D-galactosidase, have shown that the residual beta-D-galactosidase at pH 4.0 in the tissues of the affected cat is due to the neutral beta-D-galactosidase and that there is a complete deficiency of the acidic (lysosomal) beta-D-galactosidase. There is marked accumulation in all tissues and excretion in the urine of neutral oligosaccharides. Analysis of these oligosaccharides by fast-atom-bombardment mass spectrometry and g.l.c. suggests that they arise from the incomplete catabolism of N-glycans of glycoproteins. The ganglioside content of all the tissues is elevated, and it has been shown by t.l.c. that the concentration of a ganglioside fraction with a mobility similar to that of GM1 ganglioside is particularly increased. There is also some evidence of accumulation of glycosaminoglycans in the brain. The clinical symptoms, the complete deficiency of acidic beta-D-galactosidase and the storage products in visceral organs all suggest that this is a case of feline GM1-type gangliosidosis comparable with the severe infantile (Type 1) form of the disease in humans.
Active-site-serine d-alanyl-d-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes
Authors: Ghuysen J M , Frère J M , Leyh-Bouille M , Nguyen-Distèche M , Coyette J
Published: August 2015

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the ‘affinity’ of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a ‘specificity index’ to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria.
The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum
Authors: Thiyagarajah P , Lim S C
Published: August 2015

A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [(Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.
Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour
Authors: Cheeseman K H , Collins M , Proudfoot K , Slater T F , Burton G W , Webb A C , Ingold K U
Published: August 2015

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.
Regulation of rat heart cytosol 5′-nucleotidase by adenylate energy charge
Authors: Itoh R , Oka J , Ozasa H
Published: August 2015

A 5′-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5′-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.
Thyrotropin cross-links to the thyrotropin receptor through both the α and β subunits
Authors: Buckland P R , Rickards C R , Howells R D , Smith B R
Published: August 2015

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.
Molecular size of N-acetylglucosaminylphosphotransferase and α-N-acetylglucosaminyl phosphodiesterase as determined in situ in Golgi membranes by radiation inactivation
Authors: Ben-Yoseph Y , Potier M , Pack B A , Mitchell D A , Melançon S B , Nadler H L
Published: August 2015

The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N-acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N-acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes.
Purification, biochemical properties and active sites of N-acetyl-β-d-hexosaminidases from human seminal plasma
Authors: Kapur D K , Gupta G S
Published: August 2015

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.
The influence of N-acetylneuraminic acid on the properties of human orosomucoid
Authors: Friedman M L , Wermeling J R , Halsall H B
Published: August 2015

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.
Rat peritoneal mast cells release dipeptidyl peptidase II
Authors: Struckhoff G , Heymann E
Published: August 2015

Purified rat peritoneal mast cells have a 10-20-fold higher dipeptidyl peptidase II (DPP II) activity as compared with that of macrophages from the same source. Upon stimulation with the secretagogue Compound 48/80, DPP II is released from peritoneal-lavage cells and from purified mast cells, but not from purified macrophages, in a dose-dependent manner. Maximally, about one-third of the DPP II present in peritoneal-lavage cells is released. Substance P and the antigen/IgE system probably produce a similar effect. Both histamine and Zn2+, two ingredients of mast-cell granules, strongly inhibit DPP II at concentrations reported to occur in the granules. A possible role of mast-cell DPP II in the remodelling of connective tissue is discussed.
The oxidation-state-dependent ATP-binding site of cytochrome c. A possible physiological significance
Authors: Corthésy B E , Wallace C J A
Published: August 2015

Cytochrome c binds certain physiological anions that are known to modulate the biological properties of the protein, although it is not known whether this effect is fortuitous or has physiological significance. We have examined the ability of the protein and its semisynthetic analogues to associate with certain of these anions, e.g. ATP, ADP, Pi and citrate. Our results show that specific residues or clusters of residues on the surface of horse heart cytochrome c are involved in the recognition sites for these anions. We also observed that binding at one site is linked to the oxidation state of the protein.
An investigation of arterial insufficiency in rat hindlimb. An enzymic, mitochondrial and histological study
Authors: Hayes D J , Challiss R A J , Radda G K
Published: August 2015

A small animal model of arterial insufficiency has been used to investigate enzymic alterations in the gastrocnemius, plantaris and soleus muscles of the hypoperfused limb. At 7 days after induction of arterial insufficiency by unilateral femoral artery ligation, there were significant increases in the maximal activities of hexokinase, phosphorylase and 6-phosphofructokinase, whereas the activities of citrate synthase and 2-oxoglutarate dehydrogenase remained unchanged. Similar increases in hexokinase, phosphorylase and 6-phosphofructokinase were still apparent 8-10 weeks after unilateral artery ligation, although only hexokinase remained significantly higher than contralateral control values. No enhancement of oxidative enzyme activities was observed. The results are discussed in relation to the conflicting findings reported by other groups investigating enzymic adaptations in patients with arterial insufficiency.
Purification of mouse brain ornithine decarboxylase reveals its presence as an inactive complex with antizyme
Authors: Laitinen P H , Hietala O A , Pulkka A E , Pajunen A E I
Published: August 2015

Mouse brain ornithine decarboxylase (ODC) was purified to near-homogeneity by using (NH4)2SO4 precipitation and chromatography on heparin-Sepharose, pyridoxamine phosphate-agarose and DEAE-cellulose. On SDS/polyacrylamide-gel electrophoresis, the final preparation gave one protein band similar to that obtained for purified mouse kidney enzyme, corresponding to an Mr of 53.000. The overall yield of the purification exceeded about 50-fold the total activity of the enzyme in the starting material. By affinity chromatography on ODC-bound Sepharose, the extra enzyme activity was shown to originate, at least partly, from the enzyme-antizyme complex. These results demonstrate that ODC in mouse brain occurs mainly in an inactive form and is activated during purification.
The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments
Authors: Dowling L M , Crewther W G , Inglis A S
Published: August 2015

Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming subgroups of this class. The significant homologies between types I, II and III occur almost exclusively in segments of the chain that have been identified as having a coiled-coil structure together with the relatively short sections connecting these segments. The non-coiled-coil segments at the C- and N-termini show no significant homology between types, nor is homology in these segments apparent in all members of one type. Component 8c-1 does not show homology in its terminal segments with the known sequence of any other filamentin.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium
Authors: Halestrap A P , Quinlan P T , Whipps D E , Armston A E
Published: August 2015

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.
The interaction of bovine factor IX, its activation intermediate, factor IX α, and its activation products, factor IXaα and factor IXaβ, with acidic phospholipid vesicles of various compositions
Authors: Beals J M , Castellino F J
Published: August 2015

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.
Hysteretic behaviour of carnitine palmitoyltransferase. The effect of preincubation with malonyl-CoA
Authors: Cook G A , Cox K A
Published: August 2015

Continuous assays of carnitine palmitoyltransferase were used to study the hysteretic behaviour of the enzyme. When reactions were started by adding mitochondria to complete reaction mixtures, there was a lag in the assay even in the absence of malonyl-CoA. When mitochondria were preincubated with malonyl-CoA in the absence of palmitoyl-CoA, there was a greater lag period in the assay of carnitine palmitoyltransferase, but this lag was less prominent at 37 degrees C than at 30 degrees C. Preincubation of mitochondria with malonyl-CoA did not change the sensitivity of the enzyme to inhibition by malonyl-CoA.
Phorbol ester inhibits angiotensin-induced activation of phospholipase C in adrenal glomerulosa cells. Its implication in the sustained action of angiotensin
Authors: Kojima I , Shibata H , Ogata E
Published: August 2015

The present study was undertaken to determine whether an agonist-induced activation of C-kinase leads to an inhibition of phospholipase C in adrenal glomerulosa cells. When cells are treated with 100 nM-TPA (12-O-tetradecanoylphorbol 13-acetate), subsequent angiotensin (‘angiotensin II’)-induced aldosterone secretion is greatly inhibited. Treatment with TPA completely inhibits the angiotensin-induced increase in both inositol trisphosphate and the cytosolic Ca2+ concentration. The dose-response curve for TPA-induced inhibition reveals that quite a high concentration of TPA is necessary to block angiotensin action compared with that needed to stimulate aldosterone secretion. 1-Oleoyl-2-acetylglycerol has a weak inhibitory effect, whereas neither 4 alpha-phorbol 12,13-didecanoate or 4 beta-phorbol inhibits angiotensin action. When the time course of changes in inositol trisphosphate and diacylglycerol is measured, angiotensin action is sustained for up to 30 min. In addition, 100 nM-TPA added after 20 min of angiotensin addition attenuates production of both inositol trisphosphate and diacylglycerol. These results suggest that high dose of TPA inhibits angiotensin-induced activation of phospholipase C by acting, at least partly, on C-kinase, but that an inhibitory effect of TPA may be a pharmacological effect with little physiological significance in this system.
Disulphide reduction in lysosomes. The role of cysteine
Authors: Lloyd J B
Published: August 2015

Published evidence indicates that cystine-containing proteins have their disulphide bonds reduced during proteolysis in lysosomes. However, the intralysosomal accumulation of cystine in the cells of patients with cystinosis has been seen as evidence that protein cystine residues are not reduced. The data are reconcilable and fully in harmony if it is postulated that cysteine from the cytoplasm is the physiological reducing agent.
Hydrolysis of a naturally occurring β-glucoside by a broad-specificity β-glucosidase from liver
Authors: LaMarco K L , Glew R H
Published: August 2015

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.
Aromatic hydroxylation as a potential measure of hydroxyl-radical formation in vivo. Identification of hydroxylated derivatives of salicylate in human body fluids
Authors: Grootveld M , Halliwell B
Published: August 2015

Attack by .OH radicals, generated by a Fenton system, upon salicylate produces 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate as major products and catechol as a minor product. H.p.l.c. separation combined with electrochemical detection was used to identify and quantify 2,3-dihydroxybenzoate and 2,5-dihydroxybenzoate in human plasma and synovial fluid. We propose that conversion of salicylate into 2,3-dihydroxybenzoate, or of other aromatic compounds into specific hydroxylated products, may be a useful assay for .OH formation in the human body.
Free-radical chain oxidation of 2-nitropropane initiated and propagated by superoxide
Authors: Kuo C F , Fridovich I
Published: August 2015

The superoxide radical O2.-, whether produced by the xanthine/xanthine oxidase reaction or infused as KO2, solubilized by a crown ether in dry dimethyl sulphoxide, initiated a free-radical chain oxidation of anionic 2-nitropropane. Superoxide dismutase, but not catalase, inhibited oxidation of the nitroalkane. Xanthine oxidase suffered a syncatalytic inactivation, during the co-oxidation of 2-nitropropane, which was reversed by dialysis. Cyanide exacerbated this syncatalytic inactivation and rendered it irreversible. The frequently observed oxidations of nitroalkanes by flavoenzymes now need to be re-examined to clarify the extent to which O2.--initiated free-radical chain oxidation contributed to the overall nitroalkane oxidation.
Molecular forms of myeloperoxidase in human plasma
Authors: Olsen R L , Steigen T K , Holm T , Little C
Published: August 2015

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.
Ca2+ can control vascular smooth-muscle thin filaments without caldesmon phosphorylation
Authors: Marston S B
Published: August 2015

The Ca2+-dependent regulation of the activation of myosin MgATPase by vascular-smooth-muscle thin filaments involves caldesmon. This effect may be due to the direct interaction of caldesmon with a Ca2+-binding protein such as calmodulin or phosphorylation of caldesmon by a Ca2+-dependent kinase. I have found that Ca2+ switches on aorta thin filaments in less than 10 s, whereas the caldesmon in the thin filaments is phosphorylated only slowly (half-time greater than 10 min) and the maximum phosphorylation is very low (1 molecule per 7 molecules of caldesmon). I conclude that the phosphorylation of caldesmon hypothesis is untenable.
Separation of rabbit mammary-gland prolactin receptors by ion-exchange chromatography, h.p.l.c.-gel filtration and ultracentrifugation
Authors: Sakai S , Ike F , Kohmoto K , Johke T
Published: August 2015

Rabbit mammary-gland prolactin (Prl) receptors in the microsomal fraction were solubilized in 7.5 mM-Chaps) or 1% Triton X-100 and analysed by ion-exchange chromatography using DEAE-Bio-Gel A. Prl receptors in the presence of 7.5 mM-Chaps were separated into two different fractions (Fr. A and B), both of which showed identical specificity of binding to peptide hormones as those in the Chaps or Triton extract. oPrl and human growth hormone (hGH) bound to the same site, but other non-lactogenic hormones (follicle-stimulating hormone, oGH, luteinizing hormone and insulin) failed to bind to the Prl receptors. The dissociation constant (Kd) for Prl binding to the receptors in Fr. A was about 50% of those in Fr. B, suggesting that the rabbit mammary gland contains two types of Prl receptors, one with a high, and one with a low, Kd for Prl binding. A decrease in the concentration of Chaps in the column buffer to 4 mM caused aggregation of the receptors in Fr. A. H.p.l.c.-gel filtration, using Shim pack 150 and 300 columns connected in series, separated the receptor as a protein with an Mr of 74,000 +/- 4,900 (mean +/- S.D.) in the presence of 5 mM-Chaps, or of 36,800 +/- 2,100 in the presence of 7.5 mM-Chaps. Sucrose-gradient-centrifugation analysis showed that the Prl-receptor complexes in the presence of 5 mM-Chaps were sedimented between gamma-globulin and bovine serum albumin (5.56 +/- 0.22 S). As the Chaps concentration was increased to 7.5 mM, a further peak of the Prl-receptor complexes (4.01 +/- 0.23 S) appeared below ovalbumin. The present data suggest that the binding subunit causes the monomeric subunit to aggregate with itself or with another specific associated protein of similar Mr.
Structure-function relationships in the free insulin monomer
Authors: Hefford M A , Oda G , Kaplan H
Published: August 2015

The chemical properties of the functional groups of insulin were determined at a concentration (0.5 microM) where the predominant species of insulin is the free (unassociated) monomeric unit. The glycine N-terminus and the four tyrosine phenolic groups had the same properties as in the associated forms of insulin. On the other hand the lysine epsilon-amino group and the two histidine imidazole groups had substantially altered properties. Some alteration in the properties of the phenylalanine N-terminus was also observed. The reactivity-pH profile for the imidazole groups showed a second ionization with a pKa of 10.1 in addition to an ionization with a pKa of 6.8. On the basis of the X-ray-crystallographic structure of hexameric insulin the observed changes can be accounted for by disruption of monomer-monomer or dimer-dimer interactions in the associated states of insulin. It is concluded that the conformation of the monomeric unit of insulin is essentially the same in its free and associated states in solution.
How does dipyridamole elevate extracellular adenosine concentration? Predictions from a three-compartment model of adenosine formation and inactivation
Authors: Newby A C
Published: August 2015

Steady-state mathematical models are developed according to which adenosine is formed in the cytoplasm of a group of cells, arises in the extracellular space via the symmetric nucleoside transporter and is inactivated in the adenosine forming cells and after rate-limiting transport into other cell-types. Dipyridamole increases the Km and Vmax. of the transporter symmetrically with respect to influx and efflux. Models incorporating differing degrees of compartmentation are used to predict intracellular and extracellular adenosine concentration as a function of dipyridamole concentration and adenosine formation rate. The vasodilator action of dipyridamole is explained since it is predicted to elevate interstitial fluid adenosine concentrations at all rates of adenosine formation provided that washout of the interstitial compartment is restricted.
Localization and biosynthesis of polyamines in insulin-producing cells
Authors: Hougaard D M , Nielsen J H , Larsson L I
Published: August 2015

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.
Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets
Authors: Houslay M D , Bojanic D , Gawler D , O'Hagan S , Wilson A
Published: August 2015

The thrombin-stimulated GTPase activity of human platelets was additive with respect to the GTPase stimulation effected by prostaglandin E1, but not with that stimulated by adrenaline, vasopressin and platelet-activating factor (PAF). Treatment of platelet membranes with pertussis toxin partially inhibited the thrombin-stimulated GTPase, but had no effect on the vasopressin-stimulated GTPase activity, whereas cholera toxin treatment had no effect on either of these stimulated GTPase activities. Thrombin, adrenaline and PAF, but not vasopressin, inhibited the adenylate cyclase activity of isolated plasma membranes through the action of Ni only, this being inhibited by pertussis toxin. It is suggested that thrombin exerts effects through both the inhibitory guanine nucleotide regulatory protein Ni and through the putative guanine nucleotide regulatory protein, Np, involved in regulating receptor-stimulated inositol phospholipid metabolism. However, vasopressin appears to exert its effects solely through the putative Np.
Characterization of tryptophan transport in human placental brush-border membrane vesicles
Authors: Ganapathy M E , Leibach F H , Mahesh V B , Howard J C , Devoe L D , Ganapathy V
Published: August 2015

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.
Free-radical-induced formation of an 8,5′-cyclo-2′-deoxyguanosine moiety in deoxyribonucleic acid
Authors: Dizdaroglu M
Published: August 2015

Isolation and identification of a novel .OH-induced product, namely an 8,5′-cyclo-2′-deoxyguanosine moiety, in DNA and 2′-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2′-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2′-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5′R)- and (5′S)-epimers of 8,5′-cyclo-2′-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5′-cyclo-2′-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5′-cyclo-2′-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5′ of the 2′-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5′ and C-8 and subsequent oxidation of the resulting N-7-centred radical.
An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin
Authors: Calabrese L , Carbonaro M
Published: August 2015

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent ‘oxidative’ attack of proteins and lipids.
Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders
Authors: Griffiths S L , Finkelstein R A , Critchley D R
Published: August 2015

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 × 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.