bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency
Authors: Poole A R , Webber C , Reiner A , Roughley P J
Published: August 2015

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab′)2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.
Mass spectrometric detection of cross-linked fatty acids formed during radical-induced lesion of lipid membranes
Authors: Frank H , Thiel D , MacLeod J
Published: August 2015

A mass spectrometric method is described for the quantitative determination of dimers of polyunsaturated fatty acids (PUFA) formed in the hepatic endoplasmic reticulum of rats upon inhalation of tetrachloromethane. The results show that dimers account for a considerable fraction of microsomal PUFA which disappear during CCl4 metabolism. Cross-linking of the membrane lipids of the endoplasmic reticulum seems to be a significant process with respect to cell toxicity.
Neutral maltase/glucoamylase from rabbit renal cortex
Authors: Pereira B , Sivakami S
Published: August 2015

Maltase activity (EC 3.2.1.20) was solubilized from rabbit kidney brush-border membrane by using 1.0% Triton X-100 and purified 230-fold with an overall recovery of 30%. The purification procedure makes use of heat precipitation, chromatography on DE-52 DEAE-cellulose and gel filtration on Sephacryl S-300. Rabbit kidney brush border exhibited glucoamylase activity with a maltase/glucoamylase ratio of 1.5:1 to 2.0:1. During purification the maltase and glucoamylase activities behaved identically. The Mr of the complex is 590,000, and it appears to be composed of eight identical subunits linked by disulphide bridges.
Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site
Authors: Keskar S S , Srinivasan M C , Deshpande V V
Published: August 2015

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.
Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells
Authors: Lindblom A , Carlstedt I , Fransson L Å
Published: August 2015

Proteoglycans, metabolically labelled with [3H]leucine and 35SO4(2-), were isolated from the spent media and from guanidinium chloride extracts of cultured human umbilical-vein endothelial cells by using isopycnic density-gradient centrifugation, gel filtration and ion-exchange h.p.l.c. The major proteoglycan species were subjected to SDS/polyacrylamide-gel electrophoresis before and after enzymic degradation of the polysaccharide chains. The cell extract contained mainly a heparan sulphate proteoglycan that has a buoyant density of 1.31 g/ml and a protein core with apparent molecular mass 300 kDa. The latter was heterogeneous and migrated as one major and one minor band. After reduction, the apparent molecular mass of the major band increased to approx. 350 kDa, indicating the presence of intrachain disulphide bonds. The proteoglycan binds to octyl-Sepharose and its polysaccharide chains are extensively degraded by heparan sulphate lyase. The proteoglycans of the medium contained 90% of all the incorporated 35SO4(2-). Here the predominant heparan sulphate proteoglycan was similar to that of the cell extract, but was more heterogeneous and contained an additional core protein with apparent molecular mass 210 kDa. Furthermore, two different chondroitin sulphate proteoglycans were found: one 200 kDa species with a high buoyant density (approx. 1.45 g/ml) and one 100 kDa species with low buoyant density (approx. 1.3 g/ml). Both these proteoglycans have a core protein of molecular mass approx. 47 kDa.
Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase)
Authors: Deddish P A , Skidgel R A , Erdös E G
Published: August 2015

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.
Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity
Authors: Litosch I
Published: August 2015

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5′[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.
Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation
Authors: Geiger O , Görisch H
Published: August 2015

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.
Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex
Authors: Buttle D J , Kembhavi A A , Sharp S L , Shute R E , Rich D H , Barrett A J
Published: August 2015

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.
Fungal cellulase systems. Comparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and Trichoderma reesei
Authors: Claeyssens M , Van Tilbeurgh H , Tomme P , Wood T M , McRae S I
Published: August 2015

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1-4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.
Purification and catalytic properties of l-valine dehydrogenase from Streptomyces cinnamonensis
Authors: Priestley N D , Robinson J A
Published: August 2015

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4′ of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.
Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor
Authors: Murphy G , Koklitis P , Carne A F
Published: August 2015

Recombinant human tissue inhibitor of metalloproteinases (TIMP) forms complexes with high-Mr active recombinant stromelysin that are stable over long periods under physiological conditions. TIMP-stromelysin complexes could be dissociated in the presence of EDTA at pH 3, releasing free TIMP and destroying stromelysin activity. The dissociated TIMP was apparently unmodified, in contrast with other known protein inhibitors of metalloproteinases and many classes of serine-proteinase inhibitor, which are slowly cleaved.
The activities of ‘Pz-peptidase’ and ‘endopeptidase 24.15’ are due to a single enzyme
Authors: Barrett A J , Tisljar U
Published: August 2015

It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.
Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides
Authors: Baumann M , Peltonen L , Aula P , Kalkkinen N
Published: August 2015

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.
Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes
Authors: Bruce N C , Cain R B , Pieper D H , Engesser K H
Published: August 2015

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.
Concanavalin A reveals olfactory receptors which discriminate between alkane odorants on the basis of size
Authors: Polak E H , Shirley S G , Dodd G H
Published: August 2015

For certain odorants, the amplitude of the rat electro-olfactogram is reduced if the olfactory epithelium is treated with the lectin concanavalin A. When normal and cycloalkanes of one to ten carbon atoms are used as odorants at equimolar concentration, the maximum reduction in amplitude is found to correlate with the size of the stimulus molecule. This observation is consistent with the notion that concanavalin A disables an olfactory receptor molecule which normally responds to the alkyl moiety of odorants in a particular size range. That moiety may thus represent a ‘primary’ quality-determining component in odour discrimination.
Receptor-ligand interaction: a new method for determining binding parameters without a priori assumptions on non-specific binding
Authors: van Zoelen E J J
Published: August 2015

Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors.
Effects of vanadate on protein kinases in rat hepatocytes
Authors: Villar-Palasi C , Guinovart J J , Gómez-Foix A M , Rodriguez-Gil J E , Bosch F
Published: August 2015

In rat hepatocytes, vanadate modifies neither the intracellular concentration of cyclic AMP nor the -cyclic AMP/+cyclic AMP activity ratio for cyclic AMP-dependent protein kinase. Vanadate can, however, counteract the increase in cyclic AMP and the increase in the -cyclic AMP/+cyclic AMP activity ratio of cyclic AMP-dependent protein kinase induced by glucagon. On the other hand, vanadate treatment of hepatocytes can produce a time- and concentration-dependent increase in cyclic AMP- and Ca2+-independent casein kinase activity. Maximal activation at the optimal time with 5 mM-vanadate was about 70% over control. A clear relationship was observed between the activation of casein kinase and the inactivation of glycogen synthase after vanadate treatment. These results suggest that casein kinase activity may be involved in vanadate actions in rat hepatocytes.
Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular β-lactamase of Actinomadura R39
Authors: Piron-Fraipont C , Duez C , Matagne A , Molitor C , Dusart J , Frère J M , Ghuysen J M
Published: August 2015

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).
Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes
Authors: Harley C B , Menon C R , Rachubinski R A , Nieboer E
Published: August 2015

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.
Site of carbamoylation of bovine γ-II-crystallin by potassium [14C]cyanate
Authors: Martin S , Harding J J
Published: August 2015

One possible route to cataract formation may be via the carbamoylation of lens proteins due to increased concentrations of cyanate in the body resulting from uraemia associated with renal failure and with severe diarrhoea. Carbamoylation of gamma-II-crystallin, which is found in the lens core, could alter the surface charge network of the molecules, resulting in aggregation, increased light-scattering and hence cataract. We have attempted to locate the site(s) of carbamoylation in gamma-II-crystallin. gamma-II-Crystallin was isolated by gel chromatography and ion-exchange chromatography. gamma-II-Crystallin was then carbamoylated by incubation with potassium [14C]cyanate, followed by citraconylation and digestion with trypsin to give peptides that were separated by high-resolution ion-exchange chromatography. The amino acid compositions of the radioactive peptides were compared with the expected peptide composition for gamma-II-crystallin. The radioactive peptide compositions, which agreed with the theoretical peptides, all matched with the N-terminal region of gamma-II-crystallin and had in common the presence of the N-terminal glycine residue. It appears that the alpha-amino group of the N-terminal glycine was the main site of carbamoylation. This site forms part of the charge network on the surface of gamma-II-crystallin.
Ca2+-induced accumulation of pyrophosphate in mitochondria during acetate metabolism
Authors: Inoue T , Yamada T , Furuya E , Tagawa K
Published: August 2015

The mechanism of pyrophosphate (PPi) accumulation in rat liver during acetate metabolism was investigated. Perfusion of the liver with acetate in the presence of noradrenaline and glucagon induced marked accumulation of PPi (2 mumol/g of liver, 200 times that of control). In contrast, perfusion with glutamine, which generates PPi only in the cytosol, caused little accumulation of PPi, even in the presence of the two hormones. The site of PPi accumulation was shown to be the mitochondria by the finding that isolated mitochondria from the liver perfused with acetate and the hormones contained 50 nmol of PPi/mg of protein. The addition of an uncoupler to mitochondria with accumulated PPi caused gradual decrease in their PPi content, with concomitant release of a stoichiometric amount of Ca2+. Similar accumulation of PPi was observed when isolated mitochondria were incubated with acetate and Ca2+. These results show that an increase in cytosolic Ca2+ caused by the co-administration of the two hormones induced uptake of the ion into mitochondria, and that PPi accumulated in mitochondria only when it was generated in the organelles with an elevated concentration of Ca2+. High mitochondrial concentrations of Ca2+ are considered to inhibit inorganic pyrophosphatase through the formation of a stable complex, CaPPi-. Mitochondria with accumulated PPi had normal respiratory activities, and their adenine nucleotide concentrations were increased 2-fold rather than being decreased, the increases also being considered to be caused by their high concentration of Ca2+.
4-Hydroxynonenal induces a DNA-binding protein similar to the heat-shock factor
Authors: Cajone F , Salina M , Benelli-Zazzera A
Published: August 2015

By using a gel mobility assay, we have shown that treatment of HeLa cells with 4-hydroxynonenal, a major product of the peroxidation of membrane lipids and an inducer of heat-shock proteins, has the same effect as heat shock in causing the appearance of a protein which binds to the sequence of DNA specific for the induction of heat-shock genes. Lipoperoxidation and heat exposure seem to share a common mechanism of specific gene activation.
Release of penicillinase by Escherichia coli HB101 (pEAP31) accompanying the simultaneous release of outer-membrane components by Kil peptide
Authors: Aono R
Published: August 2015

The plasmid pEAP31 contains an alkaliphilic-Bacillus penicillinase gene and a colicin E1 kil gene. Escherichia coli HB101 carrying pEAP31 grown at high temperature released outer-membrane proteins, lipopolysaccharide and phosphatidylethanolamine into the culture medium. Concurrently, penicillinase that had accumulated in the periplasm of the organism was released from the cells. Phospholipase A1-A2 in the outer membrane was not activated in the organism. The results suggest that the release of accumulated periplasmic penicillinase from the producer cells was caused by partial disruption of the outer membrane mediated by the Kil peptide.
Further studies of glycosylation and intracellular transport of lactase–phlorizin hydrolase in rat small intestine
Authors: Büller H A , Rings E H M , Montgomery R K , Sasak W V , Grand R J
Published: August 2015

Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.
Fluorescence studies on the role of tryptophan in heterogeneous nuclear ribonucleoprotein particles of HeLa cells
Authors: Schenkel J , Appel I , Schwarzwald R , Bautz E K F , Wolfrum J , Greulich K O
Published: August 2015

The 40 S heterogeneous nuclear ribonucleoprotein (hnRNP) particles from HeLa cells reveal tryptophan fluorescence with a bi-exponential decay, indicating that only a few of the ‘core’ proteins contain tryptophan residues. The presence of tryptophan residues distinguishes hnRNP particles from nucleosomes, with which they otherwise share a number of properties. This difference, however, is not essential for protein-RNA binding, as the fluorescence decay remains unchanged when hnRNP particles are dissociated into protein and RNA. However, the Stern-Volmer quenching constant is doubled upon salt dissociation, i.e. tryptophan residues become more accessible to solvent. Thus tryptophan quenching is a useful parameter for monitoring protein-protein interactions in hnRNP particles.
A simplified method for calculating complex metabolic sensitivities by using matrix partitioning
Authors: Crabtree B , Collins G , Franklin M I
Published: August 2015

The matrix method for calculating the overall sensitivities (including control coefficients) of a metabolic system, described by Crabtree & Newsholme [Biochem. J. 247, 113-129 (1987)], is simplified by a preliminary partitioning of the initial matrix equation. This reduces the size of the matrix to be inverted and thereby removes a major drawback with the original method. The resulting procedure is simpler and more systematic than the alternative methods currently available, especially when the system is extensively branched.
Plant mitochondrial F1-ATPase. The presence of oligomycin-sensitivity-conferring protein (OSCP)
Authors: Horak A , Horak H , Dunbar B , Fothergill J E , Wilson S B
Published: August 2015

Purified pea (Pisum sativum) cotyledon F1-ATPase contains six subunits rather than the five usually reported for F1-ATPases. The additional 26.5 kDa (delta) subunit is shown by immunoblotting and N-terminal amino acid sequencing to be similar to bovine oligomycin-sensitivity-conferring protein (OSCP). It is concluded that the delta subunit of plant mitochondrial F1-ATPase is the plant OSCP. This OSCP subunit occurs in all mono- and di-cotyledonous species of plants tested (maize, oats, peas, potatoes, sweet potatoes and turnips).
Prolonged high intracellular free calcium concentrations induced by ATP are not immediately cytotoxic in isolated rat hepatocytes. Changes in biochemical parameters implicated in cell toxicity
Authors: Nagelkerke J F , Dogterom P , De Bont H J G M , Mulder G J
Published: August 2015

Isolated rat hepatocytes were incubated with ATP to induce high intracellular free Ca2+ concentrations as determined with the Quin-2 method. Immediately after addition of ATP, the intracellular concentration of Ca2+ rose from 200 nM to more than 2.5 microM. It stayed at this value during the first 1/2 h; the rise was absolutely dependent on extracellular Ca2+. After the first 1/2 h the Ca2+ concentration decreased to 1-2 microM (5-10 times the control value). These high intracellular free Ca2+ concentrations did not lead to an immediate loss of cell viability. Only after 2 h of incubation a substantial number of cells lost viability. This was preceded by a decrease in cellular NADH (greater than 40%) and accompanied by a sharp increase in the intracellular Ca2+ concentration. Under these conditions the NADPH concentration was not affected. Cellular GSH was decreased to 30% of the initial value, but no lipid peroxidation or protein-thiol depletion was observed. Intracellular ATP, ADP and AMP were increased in the presence of extracellular ATP. Ca2+-dependent proteases seemed not to be involved in cell death. These observations are consistent with a collapse of mitochondrial functions as a final trigger of cell death.
A simplified assay for the enzyme responsible for the attachment of myristic acid to the N-terminal glycine residue of proteins, myristoyl-CoA: glycylpeptide N-myristoyltransferase
Authors: McIlhinney R A J , McGlone K
Published: August 2015

A greatly simplified assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase (NMT) activity is described. The assay is based on the differential solubility of the acyl-peptides produced as a consequence of the NMT activity and yields results comparable with those obtained with the original assay described by Towler & Glaser [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2812-2816], which requires h.p.l.c. to determine the production of the acyl-peptides. The use of the revised assay in the preliminary steps of the purification of rat brain NMT is described, and its use in determining the fatty acid-specificity of the enzyme is illustrated. The results are shown to be comparable with those obtained with the h.p.l.c.-based assay.
Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem
Authors: Kwong T C , Sparks J D , Pryce D J , Cianci J F , Sparks C E
Published: August 2015

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.
The catalytic mechanism of the hydroxylation reaction of peptidyl proline and lysine does not require protein disulphide-isomerase activity
Authors: Myllylä R , Kaska D D , Kivirikko K I
Published: August 2015

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens. The beta subunit is known to be identical with the enzyme protein disulphide-isomerase and to possess disulphide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer. We here report that lysyl hydroxylase, a homodimer, and algal prolyl 4-hydroxylase, a monomer, do not contain detectable protein disulphide-isomerase activity. Since the hydroxylase reaction mechanisms are similar, the data suggest that the protein disulphide-isomerase activity of the vertebrate prolyl 4-hydroxylase beta subunit is unlikely to be involved in the catalytic mechanism of the hydroxylation reaction.
Differential sensitivity of anti-IgM-induced and NaF-induced inositol phospholipid metabolism to serine protease inhibitors in BAL17 B lymphoma cells
Authors: Mizuguchi J , Utsunomiya N , Nakanishi M , Arata Y , Fukazawa H
Published: August 2015

A BAL17 B lymphoma cell line bearing mu and delta chains on its surface behaves in a similar manner to normal mature B cells in terms of initial biochemical transmembrane signalling [Mizuguchi, Beaven, Ohara & Paul (1986) J. Immunol. 137, 2162-2167; Mizuguchi, Yong-Yong, Nakabayashi, Huang, Beaven, Chused & Paul (1987) J. Immunol. 139, 1054-1059]. Therefore the effects of protease inhibitors on increases in inositol phospholipid metabolism and intracellular free calcium concentration ([Ca2+]i) were examined. We show that the serine protease inhibitors Tos-Phe-CH2Cl (1-chloro-4-phenyl-3-L-tosylamidobutan-2-one-, TPCK) and Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one; TLCK) inhibit anti-IgM-mediated accumulation of inositol phosphates in a dose-dependent manner. InsP3 production induced by anti-IgM is also inhibited by pretreatment with Tos-Lys-CH2Cl or Tos-Phe-CH2Cl. Tos-Lys-CH2Cl- Tos-Phe-CH2Cl-mediated inhibition is not overcome by high concentrations of anti-IgM. Moreover, anti-IgM-mediated increases in [Ca2+]i are inhibited by pretreatment of the cells with these inhibitors. However, increases in inositol phospholipid metabolism caused by NaF, an activator of guanine-nucleotide-binding proteins (G-proteins), are approx. 10-fold more resistant to Tos-Lys-CH2Cl and Tos-Phe-CH2Cl inhibition compared with anti-IgM-induced changes. Furthermore, NaF-induced increases in [Ca2+]i are not inhibited by Tos-Lys-CH2Cl or Tos-Phe-CH2Cl pretreatment, suggesting that the inhibitors act at a step proximal to phospholipase C activation. The Tos-Lys-CH2Cl or Tos-Phe-CH2Cl treatment does not change the membrane IgM density as measured by flow cytometry, indicating that the active site of the inhibitors is distal to the membrane IgM molecule. These results indicate that serine proteases may be involved in coupling the receptor cross-linkage to G-protein.
Turnover of carnitine palmitoyltransferase mRNA and protein in H4IIE cells. Effect of cyclic AMP and insulin
Authors: Wang L , Brady P S , Brady L J
Published: August 2015

Regulation of the 68 kDa carnitine palmitoyltransferase (CPT) synthesis by (chlorophenylthio) cyclic AMP (cAMP) and insulin was studied in H4IIE cells in culture. Addition of 0.1 mM- or 1.0 mM-(chlorophenylthio) cAMP induced CPT mRNA and rate of transcription 2-4-fold by 15 min, reaching a plateau at 4-6-fold by 30 min. Addition of 5-15 nM-insulin plus 1.0 mM-cAMP suppressed the increases in transcription rate and mRNA levels occurring with cAMP alone. The t1/2 for CPT mRNA was 70-80 min and was not affected by cAMP. The t1/2 for CPT protein was 70 min, and was increased to 240 min in the presence of cAMP. The rate of CPT synthesis was also increased in the presence of cAMP. The data indicate that CPT synthesis is increased by cAMP via induction of transcription and subsequent increase in the CPT mRNA. Insulin acts to depress transcription and CPT mRNA. In addition, cAMP prolongs the t1/2 of CPT.
Formation of Nε-(γ-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells
Authors: Fesus L , Tarcsa E
Published: August 2015

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.
Two types of rate-determining step in chemical and biochemical processes
Authors: Yagisawa S
Published: August 2015

Close examination of the concept of the rate-determining step (RDS) shows that there are two types of RDS depending on the definition of ‘rate’. One is represented by the highest peak of the free-energy diagram of consecutive reactions and holds true where the rate is defined in terms of the concentration of the first reactant. The other is represented by the peak showing the maximum free-energy difference, where the free-energy difference is the height of a peak measured from the bottom of any preceding troughs, where the definition of the rate is in terms of the total reactant concentration including intermediates. There are no criteria a priori for selecting one of them.
The A-type receptor for platelet-derived growth factor mediates protein tyrosine phosphorylation, receptor transmodulation and a mitogenic response
Authors: Hammacher A , Nistér M , Heldin C H
Published: August 2015

A clonal human glioma cell line, U-343 MGa 31L, which expresses the A-type but not the B-type receptor for platelet-derived growth factor (PDGF), was used in a functional study of the A-type receptor. PDGF-AA induced, in a dose- and time-dependent manner, phosphorylation on tyrosine residues of the receptor in metabolically labelled cells. The optimal dose was around 30 ng/ml; at 100 ng/ml, phosphorylation was maximal at 15 min and had almost returned to the control level after 60 min. The phosphorylation on tyrosine residues of the PDGF A-type receptor was stimulated by PDGF-AA, PDGF-AB and PDGF-BB; these isoforms also stimulated [3H]thymidine incorporation into U-343 MGa 31L cells. In addition, activation of the A-type PDGF receptor induced transmodulation of the epidermal growth factor receptor.