bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites
Authors: Willenbrock F , Brocklehurst K
Published: August 2015

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.
Effect of benzyl γ-d-xyloside on the biosynthesis of chondroitin sulphate proteoglycan in cultured human monocytes
Authors: Kolset S O , Ehlorsson J , Kjellén L , Lindahl U
Published: August 2015

Monocytes isolated from human blood differentiate into macrophage-like cells when maintained in vitro for 3-5 days on plastic or glass culture dishes. In the process the cells display characteristic morphological changes, and in addition, a transition in glycosaminoglycan biosynthesis, from the production of chondroitin 4-sulphate to the formation of a polysaccharide containing 20% 4,6-disulphated disaccharide units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. Cells were incubated with inorganic [35S]sulphate on day 1 or day 6 in culture, in the presence or absence of benzyl beta-D-xyloside, and labelled polysaccharide was isolated from the culture medium. In the presence of xyloside, the secretion of proteoglycans (90% galactosaminoglycan) was inhibited in a dose-dependent fashion and replaced by release of single polysaccharide chains, the size of which decreased with increasing dose of xyloside. The single polysaccharide chains produced on day 6 in the presence of 0.5 mM-xyloside showed the same proportion of disulphated disaccharide units as did the corresponding control material. Day-1 polysaccharide contained negligible amounts of this component, irrespective of the presence or absence of xyloside. It is concluded that the regulatory mechanism that induces ‘oversulphation’ during the differentiation process operates independently of any association between the polysaccharide chains and the core protein. Moreover, cells maintained in the presence of 0.5 mM-xyloside throughout a 6-day culture period showed the same morphological change, indicative of differentiation into macrophage-like cells, as did untreated control cells. The xyloside did not significantly affect the cytotoxicity of the monocytes, or of the differentiated macrophage-like cells, toward tumour cells.
The nucleotide sequence of Scenedesmus obliquus chloroplast elongator methionine-accepting tRNA
Authors: McCoy J M , Keene N M , Jones D S
Published: August 2015

The chloroplast elongator methionine-accepting tRNA from the green alga Scenedesmus obliquus was purified and and its nucleotide sequence determined. The sequence does not bear close homology to higher-plant chloroplast tRNAMetm species, nor to the chloroplast tRNAMetm species from Euglena gracilis. This tRNA is unique among all tRNAs in having pseudouridine at position 8, and this has implications with regard to the proposed interaction of aminoacyl-tRNA synthetases with their cognate tRNAs at this position. The primary sequence of the tRNA is: pG-C-C-U-G-C-U-psi-A-G-C-U-C-A-G-U-D-Gm- G-C-C-A-G-A-G-C-A-U-C-C-G-psi-psi-U-C-A-U-m2A-C-G-C-G-G-A-A-A- m7G-D-C-A-C-U-A-G-T-psi-C-G-A-A-U-C-U-A-G-U-A-G-C-A-G-G-C-A-C-C-A-OH.
Interactions of cephalosporins with the Streptomyces R61 dd-transpeptidase/carboxypeptidase. Influence of the 3′-substituent
Authors: Faraci W S , Pratt R F
Published: August 2015

The influence and fate of the 3′-substituent of a series of cephalosporins on their interaction with the DD-transpeptidase/carboxypeptidase of Streptomyces R61 were investigated. Good 3′-leaving groups are eliminated from the acyl-enzyme before deacylation, and hence cephalosporins with such substituents generate a common covalent intermediate. The rate of this elimination reaction in the two cases studied in detail was much lower than from the corresponding cephalosporoates in free solution. Cephalosporins without good 3′-leaving groups yield acyl-enzymes that appear to hydrolyse, leading to enzyme re-activation, faster than those from cephalosporins with leaving groups.
Differential tissue expression of the glutathione transferase multigene family
Authors: Pemble S E , Taylor J B , Craig R K , Ketterer B
Published: August 2015

The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.
The role of polyphosphoinositides and their breakdown products in A23187-induced release of arachidonic acid from rabbit polymorphonuclear leucocytes
Authors: Meade C J , Turner G A , Bateman P E
Published: August 2015

Stimulation of rabbit polymorphonuclear leucocytes with A23187 causes phospholipase C mediated breakdown of polyphosphoinositides, as evidenced by accumulation of [3H]inositol-labelled inositol bisphosphate and inositol trisphosphate. At the same time the polyphosphoinositides and the products of their breakdown, diacylglycerol and phosphatidic acid, label rapidly with radioactive arachidonic acid. Enhancement of polyphosphoinositide labelling is not as great as enhancement of diacylglycerol or phosphatidic acid labelling, suggesting additional early activation of a second independent synthetic pathway to the last named lipids. Experiments using double (3H/14C) labelling, to distinguish pools with different rates of turnover, suggest the major pool of arachidonic acid used for synthesis of lipoxygenase metabolites turns over more slowly than arachidonic acid in diacylglycerol, but at about the same rate as arachidonic acid esterified in phosphatidylcholine or phosphatidylinositol. Further, when cells are prelabelled with [14C]arachidonic acid, then stimulated for 5 min, it is only from phosphatidylcholine, and to a lesser extent phosphatidylinositol, that radiolabel is lost. Release of arachidonic acid is probably via phospholipase A2, since it is blocked by the phospholipase A2 inhibitor manoalide. The absence of accumulated lysophosphatides can be explained by reacylation and, in the case of lysophosphatidylinositol, deacylation. The importance of phospholipase A2 in phosphatidylinositol breakdown contrasts with the major role of phospholipase C in polyphosphoinositide hydrolysis. Measurements of absolute free fatty acid levels, as well as studies showing a correlation between production of radiolabelled hydroxyeicosatetraenoic acids and release of radiolabel from the phospholipid pool, both suggest that hydrolysis of arachidonic acid esterified into phospholipids is the limiting factor regulating formation of lipoxygenase metabolites. By contrast with A23187, fMet-Leu-Phe (a widely used polymorphonuclear leucocyte activator) is a poor stimulant for arachidonic acid release unless a ‘second signal’ (e.g. cytochalasin B, or a product of A23187-stimulated cells) is also present. In the presence of cytochalasin B, fMet-Leu-Phe, like A23187, stimulates release of radiolabelled arachidonic acid principally from phosphatidylcholine.
Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain
Authors: Mani R S , Kay C M
Published: August 2015

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.
Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface
Authors: Ward D McV , Kaplan J
Published: August 2015

Incubation of serum-growth HeLa cells in serum-free medium causes a rapid (t1/2 3 min) 30-60% decrease in the binding of 125I-diferric transferrin to the cell surface. Addition of fetal bovine serum to cells in serum-free medium results in a rapid (t1/2 3 min) and concentration-dependent increase in binding activity. The loss or gain in ligand binding is a result of changes in surface receptor number rather than an alteration in ligand-receptor affinity. A variety of hormones (insulin, insulin-like growth factor, interleukin 1 and platelet-derived factor) were found to mimic the effect of serum on receptor number. The alteration in surface receptor number was found to be calcium-dependent. Changes in surface receptor number were independent of either receptor biosynthetic rate or the absolute cellular content of receptors. The effect of insulin or serum on Hela cell transferrin receptor distribution was unaffected by the presence of transferrin, demonstrating that receptor distribution in this cell type is ligand-independent. The ability of serum or insulin to modify surface transferrin receptor number was also observed in mouse L-cells, human skin fibroblasts, and J774 macrophage tumour cells. However, transferrin receptors on K562 and Epstein-Barr virus-transformed human lymphoblasts were unaltered by these agents. The quantities of receptors whose distribution is predominantly on the surface (i.e. epidermal growth factor or low density lipoprotein receptor) were unaltered by addition of the mitogenic agents. These results extend our previous studies [H.S. Wiley & J. Kaplan (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7456-7460] demonstrating that mitogenic agents can induce redistribution of receptor pools in selected cell types.
Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas
Authors: Taylor C W , Merritt J E , Putney J W , Rubin R P
Published: August 2015

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.
Intermediate Mr cytosolic components potentiate hepatic 5′-deiodinase activation by thiols
Authors: Sawada K , Hummel B C W , Walfish P G
Published: August 2015

The role in the activation of microsomal 5′-deiodinase (5′-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5′-DI activity, could not stimulate microsomal 5′-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated ‘deiodination factor B’ (DFB), operates as an efficient intermediary to enhance activation of microsomal 5′-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5′-DI thiol (ESI) to its active state (ESH).
Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation
Authors: D'Amore T , Cheung M O , Duronio V , Lo T C Y
Published: August 2015

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).
PAF-acether (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine)-induced fibrinogen binding to platelets depends on metabolic energy
Authors: Kloprogge E , Hasselaar P , Akkerman J W N
Published: August 2015

A combination of CN- and 2-deoxy-D-glucose decreases the binding of fibrinogen to platelets stimulated with PAF-acether (1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine). Decreased binding is found after pretreatment with metabolic inhibitors, thereby lowering the energy content before stimulation as well as at various stages after stimulation of undisturbed cells. Binding and ATP hydrolysis occur in parallel, suggesting tight coupling between both phenomena. Energy appears to be predominantly required for exposure and maintenance of accessible binding sites, whereas the interaction between fibrinogen and the exposed sites does not depend on metabolic energy.
The ability of salts to inhibit the reaction between periodate anions and ovotransferrin
Authors: Hsuan J J
Published: August 2015

The reaction between periodate anions and apo-ovotransferrin results in the rapid abolition of the iron-binding ability of the protein and the loss of approximately 4 mol of tyrosine/mol of protein. The degree of inhibition exerted by a variety of salts on the rate of this reaction is found to be inconsistent with the lyotropic series and suggests the existence of a complex anion-binding site in the apoprotein. The existence of this site may explain the action of periodate anions on ovotransferrin.
Structural characterization and exposure of aromatic residues in epidermal growth factor from the rat
Authors: Mayo K H , Schaudies P , Savage C R , De Marco A , Kaptein R
Published: August 2015

Aromatic amino acid residues in epidermal growth factor (EGF) isolated from the rat have been investigated by proton n.m.r. and nuclear Overhauser methods at 500 MHz and by photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) experiments at 360 MHz. Rat EGF contains six aromatic residues, i.e. one histidine and five tyrosine residues. pH titration data allow identification of the histidine imidazole ring protons, whereas two-dimensional n.m.r. correlated spectroscopy establishes connectivities between tyrosine ring (2,6) and (3,5) proton resonances. Photo-c.i.d.n.p. data give evidence for solvent exposure of the one histidine and the five tyrosine residues in rat EGF. Nuclear Overhauser experiments and pH titration data suggest proximity relationships among four of the tyrosine residues and the histidine residue. These data indicate the presence of a clustered, aromatic, structural domain on the protein surface and may provide a clue to the understanding of the functional structure of EGF.
The structure of the hepatic insulin receptor and insulin binding
Authors: Haynes F J , Helmerhorst E , Yip C C
Published: August 2015

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.
Structural studies of a carbohydrate-containing immunoglobulin-λ-light-chain amyloid-fibril protein (AL) of variable subgroup III
Authors: Holm E , Sletten K , Husby G
Published: August 2015

The amino acid sequence of the variable region of a carbohydrate-containing amyloid-fibril protein MOL of immunoglobulin-light-chain type (AL) was elucidated. The sequence determination involved cleaving the protein with CNBr, BNPS-skatole, thermolysin and trypsin. The sequenced protein consisted of about 130 amino acid residues; however, gel-filtration and N-terminal analysis studies revealed AL proteins ranging in Mr from about 10,000 to 25,000. The oligosaccharide chain was found to be bound in the hypervariable region. By sequence homology to other lambda chains the AL protein MOL was shown to be of the V lambda III subgroup.
Guanine nucleotide binding protein involved in muscarinic responses in the pig coronary artery is insensitive to islet-activating protein
Authors: Sasaguri T , Hirata M , Itoh T , Koga T , Kuriyama H
Published: August 2015

In an attempt to identify the nature of guanine nucleotide binding protein(s) (G-protein) involved in the acetylcholine (ACh)-induced (muscarinic) response of pig coronary-artery smooth muscle, we studied the effect of ADP-ribosylation of specific membrane protein(s) catalysed by islet-activating protein (IAP; pertussis toxin). The ACh-stimulated and guanine nucleotide-dependent activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE), assessed by the production of inositol 1,4,5-trisphosphate (IP3) from exogenously applied PIP2, were not modified, in either IAP-treated or non-treated cell homogenates used as the enzyme source. In intact tissues, pretreatment with up to 100 ng of IAP/ml inhibited neither the ACh-induced decrease in the amount of inositol phospholipids nor the increase in the amounts of phosphatidic acid and of inositol phosphates. IAP treatment increased the amount of cyclic AMP accumulated by isoprenaline. These observations suggest that G-protein which couples the muscarinic receptor to PIP2-PDE is insensitive to IAP. Such being the case, the nature of this protein(s) probably differs from that required for the regulation of adenylate cyclase activities (Ni or Gi).
The development of cytochrome b-245 in maturing human macrophages
Authors: Garcia R C , Cross A R , Segal A W
Published: August 2015

Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.
Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase
Authors: Reiss N , Kanety H , Schlessinger J
Published: August 2015

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.
Purification and characterization of 3-dehydroquinase from Escherichia coli
Authors: Chaudhuri S , Lambert J M , McColl L A , Coggins J R
Published: August 2015

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.
Isolation and identification of uridine(5′)-diphospho(1)-2,3-diacetamido-2,3-dideoxy-α-d-glucopyra nuronic acid from Pseudomonas aeruginosa P1-III
Authors: Okuda S , Murata S , Suzuki N
Published: August 2015

A new uridine nucleotide was isolated from Pseudomonas aeruginosa P1-III (Habs serotype 5). On the basis of 13C-n.m.r. and p.m.r. spectroscopy, mass spectrometry, i.r.-absorption spectroscopy and circular dichrometry, the structure of the new compound was unequivocally identified as uridine(5′)-diphospho(1)-2,3-diacetamido-2,3-dideoxy-alpha-D-gl ucopyranuronic acid.
Modification of human haemoglobin with glucose 6-phosphate enhances tetramer-dimer subunit dissociation
Authors: Valdes R
Published: August 2015

Studies using equilibrium gel-permeation chromatography demonstrate that formation of the covalent adduct of D-glucose 6-phosphate (G6P) with human haemoglobin promotes dissociation of the haemoglobin tetramer into its component alpha beta dimer pairs [Kdoxy = 2.57 × 10(-6) versus Kdoxy (G6P) = 11.22 × 10(-6) M-haem]. On the other hand, Kd for glucosylated haemoglobin is identical with those of the O2- and CO-liganded forms of intact haemoglobin A0. These data are consistent with the phosphate moiety alone being responsible for a 4.5-fold increase in the tetramer-to-dimer apparent Kd. This suggests the glucose 6-phosphate moiety does not bind to the same sites on haemoglobin as do the free organic phosphates, as suggested by ligand-binding kinetics data or structural analysis. My study presents a working model for studying changes in protein subunit assembly as altered by protein phosphorylations.
Cat pancreatic eicosapeptide and its biosynthetic intermediate Conservation of a monobasic processing site
Authors: Nielsen H V , Gether U , Schwartz T W
Published: August 2015

Pancreatic eicosapeptide is synthesized together with the hormone pancreatic polypeptide in a common precursor in the major endocrine cell type of the duodenal pancreas. This processing has been previously demonstrated in man and in the dog. In the present study the cat pancreatic eicosapeptide and a C-terminally extended form of this were isolated and characterized from acid/ethanol extracts of pancreas by gel filtration and reverse-phase h.p.l.c. The sequence homology in the C-terminal part of the eicosapeptides from different species was shown to continue to the other side of the monobasic cleavage site in the extended intermediate form, whereas the end of the extension differed both in chain length and amino acid sequence. It is concluded that the processing site in the intermediate form of the pancreatic eicosapeptide is an example of a proline-directed monobasic cleavage site that has been conserved during evolution.
Rat heparins A study of the relative sizes and antithrombin-binding characteristics of heparin proteoglycans, chains and depolymerization products from rat adipose tissue, heart, lungs, peritoneal cavity and skin
Authors: Horner A A
Published: August 2015

35S-labelled heparins were recovered from adipose tissue, hearts, lungs, peritoneal cavities and skins of rats given H2(35)SO4. Their purification involved incubation with Pronase, precipitation with cetylpyridinium chloride in 1.0 M-NaCl, gradient elution from DEAE-Sephacel and incubation with chondroitinase ABC. Each product was divided into proteoglycan and ‘depolymerization products’ fractions by gel filtration on Bio-Gel A-15m. Heparin chains were released from a portion of each proteoglycan fraction by beta-elimination with NaOH. Proteoglycans, chains and depolymerization products were separated by gradient elution from a column of antithrombin-agarose into fractions with no affinity, low affinity and high affinity for antithrombin. The relative sizes of the products were determined by gel filtration on columns of Bio-Gel A-50m, A-15m, A-1.5m and A-0.5m. Skin was the major source of heparin and contained the largest proteoglycans and the lowest proportion of depolymerization products. Lungs contained the smallest proteoglycans, the smallest depolymerization products and the highest proportion of depolymerization products. The highest proportions of proteoglycans, chains and depolymerization products with high affinity for antithrombin were found in adipose tissue. The lowest proportions of each of these fractions were found in the peritoneal cavity. The data suggest that there was relatively little biosynthesis of sites with high affinity for antithrombin in peritoneal-cavity mast cells and that heparin catabolism was most active in lungs. Each source of heparin was unique with respect to both biosynthesis and subsequent breakdown of its proteoglycans.
A suitable parameterization of the Michaelis-Menten enzyme reaction
Authors: Ratkowsky D A
Published: August 2015

It is shown here that a suitable form for estimation and inference using the Michaelis-Menten [(1913) Biochem Z. 49, 333-369] model for simple enzymic reactions is one in which the two parameters appear in the denominator of the equation. In this form, convergence to the least-squares estimates using the Gauss-Newton method [see Kennedy & Gentle (1980) Statistical Computing, Marcel Dekker, New York] is virtually ensured, or, as the model in this form is a member of the class of ‘generalized linear models’, it may be fitted by packages such as those of Rothamsted Experimental Station [(1977) GENSTAT (A General Statistical Program), Rothamsted Experimental Station, Harpenden] and the Numerical Algorithms Group [(1978) GLIM (Generalised Linear Interactive Modeling), Numerical Algorithms Group, Oxford]. Furthermore, the parameters-in-denominator principle is readily extended to more complicated catalytic models. With all parameters in the denominator, the least-squares estimators are close to being unbiased and normally distributed, whereas severe bias and non-normality may result from use of the standard formulations.
Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors Evidence for the involvement of a guanine nucleotide regulatory protein
Authors: Bojanic D , Fain J N
Published: August 2015

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.
Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage
Authors: McQuillan D J , Handley C J , Campbell M A , Bolis S , Milway V E , Herington A C
Published: August 2015

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.
Conversion of human placental alkaline phosphatase from a high Mr form to a low Mr form during butanol extraction. An investigation of the role of endogenous phosphoinositide-specific phospholipases
Authors: Malik A S , Low M G
Published: August 2015

Alkaline phosphatase in a wide range of tissues has been shown to be anchored in the membrane by a specific interaction with the polar head group of phosphatidylinositol. It has previously been suggested that the production of low Mr alkaline phosphatase during the commonly used butanol extraction procedure may result from the activation of an endogenous phosphoinositide-specific phospholipase C which removes the 1,2-diacylglycerol responsible for membrane anchoring. This conversion process was investigated in greater detail with human placenta used as the source of alkaline phosphatase. Mr and hydrophobicity of the alkaline phosphatase were determined by gel filtration on TSK-250 and partitioning in Triton X-114, respectively. Alkaline phosphatase extracted from human placental particulate fraction with butanol at pH 5.4 or released by incubation with Staphylococcus aureus phosphatidylinositol-specific phospholipase C produced a form of alkaline phosphatase of Mr approx. 170,000 and relatively low hydrophobicity. By contrast, the butanol extract prepared at pH 8.3 was an aggregated form of Mr approx. 600,000 and was relatively hydrophobic. The effect of a variety of inhibitors and activators on the amount of low Mr alkaline phosphatase produced during butanol extraction revealed that it was a Ca2+- and thiol-dependent process. Proteinase inhibitors had no effect. [3H]Phosphatidylinositol hydrolysis by the particulate fraction, unlike low Mr alkaline phosphatase production, was relatively sensitive to heat inactivation, indicating that the phosphoinositide-specific phospholipases C from cytosol and lysosomes were unlikely to be responsible for conversion. A butanol-stimulated activity which removed the [3H]myristic acid from the variant surface glycoprotein ([3H]mfVSG) of Trypanosoma brucei was detectable in the human placental particulate fraction. Since this activity was acid active, Ca2+- and thiol-dependent and relatively heat stable, it may be the same as that responsible for production of low Mr alkaline phosphatase. The only 3H-labelled product identified was phosphatidic acid, suggesting that the [3H]mfVSG-cleaving activity is a phospholipase D. These data strongly support the proposal that production of low Mr alkaline phosphatase during butanol extraction is an autolytic process occurring as the result of an endogenous phospholipase. However, they also suggest that the lysosomal and cytosolic phosphoinositide-specific phospholipases C that have previously been described in many mammalian tissues are not responsible for this process.
Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas
Authors: Bartelt D C , Wolff D J , Scheele G A
Published: August 2015

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.
The exponential model for a regulatory enzyme An interpretation of the linear free-energy relationship
Authors: Ainsworth S
Published: August 2015

A physical mechanism is suggested to explain the linear free-energy relationship employed in the exponential model for a regulatory enzyme [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The interpretation depends on the assumption that the structure of the enzyme changes in proportion to its saturation by substrate but at a rate that is low compared with the rates of the association-dissociation reactions of the enzyme-substrate system.
Histone acetylation in chicken erythrocytes. Estimation of the percentage of sites actively modified
Authors: Zhang D , Nelson D A
Published: August 2015

Within the N-terminal regions of the DNA-bound histone, 26 lysine residues per nucleosome may be acetylated and deacetylated. In the present paper the percentage of these residues actively acetylated and deacetylated in chicken erythrocytes was measured. This percentage is estimated as 3.7% in chicken immature, and 2.1% in chicken mature, erythrocytes. In metabolically active, dividing, cells one would predict that, after a few generations, each site would at some point in time be modified. We conclude that, in the relatively inactive immature chicken erythrocyte, no more than 1-2% of the genome is composed of dynamically acetylated and deacetylated histone, this percentage decreasing with cell maturity. The active histone acetylation and deacetylation may be confined to transcriptionally active or potentially active erythrocyte domains.
Chromatographic resolution of chicken phosvitin. Multiple macromolecular species in a classic vitellogenin-derived phosphoprotein
Authors: Wallace R A , Morgan J P
Published: August 2015

Chicken phosvitin was prepared from egg yolk by a variety of published methods, including a modification of our own original procedure. Yolk granules and all phosvitin preparations have been previously found to contain major phosphoproteins at Mr 40,000 and 33,000 and minor satellite components when electrophoresed on polyacrylamide gradient gels and stained with Stains-all. However, only our current preparation contained three additional phosphoproteins (Mr 18,000, 15,000 and 13,000) that are also present in yolk granules. Our current phosvitin preparation also appeared to have additional components when compared with other preparations by size-exclusion and anion-exchange chromatography. Particularly complex but entirely reproducible patterns were obtained by hydrophobic-interaction chromatography. However, a cross-referencing of fractions eluted by size-exclusion chromatography to the other procedures employed, including gel electrophoresis, reinforced the notion that unfractionated chicken phosvitin contains at least five major components, designated B, C, E1, E2 and F for the Mr 40,000, 33,000, 15,000, 18,000, and 13,000 phosphoproteins, respectively. Stoichiometric considerations lead us to suggest that vitellogenin I gives rise to phosvitins C and F, vitellogenin II gives rise to phosvitin B, and vitellogenin III gives rise to either phosvitin E1 or E2, but not both. Thus, a fourth, as yet undetected, vitellogenin may exist for the chicken.
A triple-binding-domain model explains the specificity of the interaction of a sphingolipid activator protein (SAP-1) with sulphatide, GM1-ganglioside and globotriaosylceramide
Authors: Wynn C H
Published: August 2015

The conformations of the neutral glycosphingolipid, globotriaosylceramide, and of the methyl ester of GM1-ganglioside have been predicted by energy-minimization techniques and compared with those previously obtained for GM1- and GM2-ganglioside. A triple-binding-domain model is put forward to explain known specificities of binding between these glycosphingolipids and activator proteins. This model suggests that hydrophobic interactions, electrostatic interactions and hydrogen-bonding between sugar residues are important. The model is discussed in relation to previous studies on the effect of chemical modification of glycosphingolipids on their ligand properties.
Collagen metabolism in fibrotic liver. Effects of concanavalin A and aggregated myeloma immunoglobin G
Authors: Takahashi S , Lee M J
Published: August 2015

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.