bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrolysis of monomolecular layers of synthetic sphingomyelins by sphingomyelinase of Staphylococcus aureus
Authors: Yedgar S , Cohen R , Gatt S , Barenholz Y
Published: August 2015

The enzymic hydrolysis of three synthetic sphingomyelins, spread as monomolecular films at the air/water interface by purified Staphylococcus aureus sphingomyelinase was studied. Each of the three sphinomyelins (DL-erythro-N-palmitoyl-, -N-stearoyl- and -N-lignoceryl-sphingosylphosphocholine) has an optimal activity-dependent surface pressure or concentration curve. The optimal surface pressure as well as the optimal surface density for hydrolysis was different for each of the three substrates. This optimum coincides with the liquid-condensed/liquid-expanded phase transition for each of the sphingomyelins. At initial surface pressures (pi 0) below the optimum, reaction rates are controlled mainly by surface density of the substrate; above the optimal pi 0, reaction rates decrease with increasing surface pressure. The difference between the three synthetic sphingomyelins are explained by the variation in the degree of asymmetry between their two paraffinic chains.
The identification of protected tyrosine residues in iron-ovotransferrin
Authors: Williams J
Published: August 2015

Tetranitromethane reacts with essentially all 21 tyrosine residues of iron-free ovotransferrin. In iron-ovotransferrin, 7 mol of tyrosine/mol of protein are unreactive. Peptides containing the unreactive tyrosine residues were isolated from digests of nitrated iron-ovotransferrin. By comparing the structures of the peptides with the amino acid sequence of ovotransferrin it is found that there are ten protected residues occupying positions 42, 82, 92, 188, 319, 415, 431, 521 and 524 in the polypeptide chain. The problem of identifying the tyrosine residues that form bonds with the metal atoms is discussed.
Association of proacrosin with phospholipid membranes
Authors: Straus J. William , Polakoski Kenneth L.
Published: August 2015

Proacrosin, the zymogen precursor of acrosin, was shown to associate with anionic phospholipid membranes through apparent electrostatic charge interactions. This association was diminished by elevated cation concentrations and was dependent on membrane composition, as shown both by direct binding assays and by following the phospholipid stimulation of conversion of proacrosin into acrosin.
A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding
Authors: Yang Feng-Ming , Feng Shan-Jung , Lai Tsai-Chun , Hu Meng-Chun
Published: August 2015

As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation.
Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis
Authors: Garcia-Fabiani Maria B. , Montanaro Mauro A. , Lacunza Ezequiel , Cattaneo Elizabeth R. , Coleman Rosalind A. , Pellon-Maison Magali , Gonzalez-Baro Maria R.
Published: August 2015

Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on–off expression pattern responds predominantly to epigenetic modifications.
Comparative study of the carbohydrate moieties of normal and pathological human immunoglobulins M.
Authors: Cahour A , Debeire P , Hartmann L , Montreuil J
Published: August 2015

The well-known heterogeneity of normal and pathological immunoglobulins M was investigated in a study involving the characterization of their carbohydrate moieties. Oligosaccharide units were released from the native molecule by hydrazinolysis, and they were fractionated by affinity chromatography on a concanavalin A-Sepharose column to yield separate N-acetyl-lactosaminic-type and oligomannosidic-type structures. Further identification of these oligosaccharides was attempted by t.l.c. on silica gel and by determination of their monosaccharide compositions. A comparative study of the oligosaccharide units belonging to each population of immunoglobulin M was possible. Similarities were found in the occurrence of both types of oligosaccharide structures, and, in addition, a common double heterogeneity could be demonstrated for N-acetyl-lactosaminic-type structures: they could be resolved by affinity chromatography into bi-, tri- and tetra-antennary structures, and they also showed differences in N-acetylneuraminic acid content. Though some variations were observed in the exact composition of the oligosaccharide units within each population, it was possible to consider a representative oligosaccharide-unit composition of normal immunoglobulin M as a standard for comparison. On this basis a predominance of multi-antennary structures was observed in the more glycosylated pathological immunoglobulins M (10% carbohydrate content), whereas oligomannosidic structures were increased in pathological immunoglobulins M with a lower content of carbohydrates (7%). These variations are thought to reflect differences in the biosynthetic processing pathway of the carbohydrate units of the pathological immunoglobulins M or the enhanced expression of a molecular clone.
Changes in cell-surface fucose-containing glycopeptides and adhesion of cultured intestinal epithelial cells as a function of cell density
Authors: Sasak W , Quaroni A , Herscovics A
Published: August 2015

Confluent cultured intestinal epithelial cells displayed greater adhesion to the substratum than did subconfluent cells. Subconfluent and confluent cells were labelled with [3H]fucose for 24h and the cell-surface components were released by mild Pronase treatment. After extensive Pronase digestion, cell-surface and cell-residue glycopeptides were fractionated on Bio-Gel P-6. The cell surface contained a higher proportion of lower-molecular-weight glycopeptides than the residue. No significant difference in elution pattern was found between total cell-surface glycopeptides of subconfluent and confluent cells. However, confluent cells contained almost twice as much [3H]-fucose-labelled glycopeptides that were bound to concanavalin A-Sepharose and were subsequently eluted with 20mM-methyl alpha-D-glucopyranoside as subconfluent cells. When the bound glycopeptides were chromatographed on Bio-Gel P-6, it was found that confluent cells contained a larger proportion of lower-molecular-weight glycopeptides than subconfluent cells. This difference in size was eliminated after treatment of glycopeptides with sialidase. When growth of subconfluent cells was inhibited with a non-toxic concentration of retinoic acid, no significant effect on the elution pattern of [3H]fucose-labelled glycopeptides was observed on either Bio-Gel P-6 or concanavalin A-Sepharose. No significant difference was found in the total [3H]fucose-labelled glycoproteins from subconfluent and confluent cells by two-dimensional gel electrophoresis. It is suggested that the differences in [3H]fucose-labelled glycopeptides between subconfluent and confluent cells are cell-density-dependent rather than growth-dependent, and that these differences are likely to result from some changes in glycosylation mechanism(s). Furthermore, the differences in cell-surface glycopeptides may be related to the changes in the adhesion of the cells to the substratum.
Electrostatic interactions in the heparin-enhanced reaction between human thrombin and antithrombin
Authors: Petersen L C , Jørgensen M
Published: August 2015

Binding of heparin to thrombin is monitored by means of an aqueous two-phase partition system, and binding of heparin to antithrombin is monitored by means of heparin induced enhancement of the intrinsic fluorescence of the protein. Both types of binding are studied at various electrolyte compositions of the medium. Heparin is displaced from thrombin at lower concentrations of electrolyte than those necessary for its displacement from antithrombin. K+ is more efficient than Na+, which is again more efficient than Li+ in displacing heparin from these proteins. The kinetics of the reaction between thrombin and antithrombin in the presence of heparin were studied by using an assay where synthetic peptide substrate is present in the reaction mixture during the reaction between proteinase and inhibitor. The kinetics are studied at various electrolyte compositions of the medium and the results are compared with those obtained from the binding studies performed under similar conditions. The results are consistent with a model where binding of heparin to antithrombin causes enhancement of the reaction rate, and where this enhancement is abolished again when additional binding of heparin to thrombin takes place on further addition of heparin.
Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization
Authors: Ohtake H , Suyemitsu T , Koga M
Published: August 2015

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.
Proteoglycans of human gingival epithelium and connective tissue
Authors: Bartold P M , Wiebkin O W , Thonard J C
Published: August 2015

Proteoglycans extracted from separated specimens of healthy human gingival epithelium and from connective tissue have been purified. The epithelial proteoglycans fractionated as a single included peak on Sepharose 4B-CL and contained heparan sulphate and dermatan sulphate glycosaminoglycans. The connective-tissue proteoglycans separated into three major populations on Sepharose 4B-CL, one of which was excluded from this gel under associative conditions (0.5 M-sodium acetate, pH 7.4). Subsequent fractionation of the excluded material under dissociative conditions (4 M-guanidinium chloride/0.05 M-sodium acetate, pH 7.4) revealed an absence of any aggregate formation of molecules within this population. The connective-tissue proteoglycans contained heparan sulphate, dermatan sulphate and chondroitin 4-sulphate, the proportions of which varied with the molecular size of the proteoglycans. Amino acid analysis of the protein cores of gingival-epithelial and connective-tissue proteoglycans revealed differences that were similar to the differences described between other types of proteoglycans such as those from skin.
Asymmetry of lipid organization in cholinergic synaptic vesicle membranes
Authors: Michaelson D M , Barkai G , Barenholz Y
Published: August 2015

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.
A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore
Authors: Chang J Y , Knecht R , Braun D G
Published: August 2015

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4′-iodoacetamide and 4-dimethylaminoazobenzene-4′-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.
88MHz 113Cd-n.m.r. studies of native rat liver metallothioneins
Authors: Nicholson J K , Sadler P J , Cain K , Holt D E , Webb M , Hawkes G E
Published: August 2015

Well-resolved 113Cd-n.m.r. spectra of 113Cd-induced rat liver metallothioneins 1 and 2 are obtainable even at 88 MHz (9.4T). The line-widths of resonances are not dominated by chemical-shift-anisotropy relaxation. The increased spectral dispersion will significantly aid the study of the native (Cd- and Zn-containing) metallothioneins.
Preparation of the alkali and P light chains of chicken gizzard myosin. Amino acid sequence of the alkali light chain
Authors: Grand R J A , Perry S V
Published: August 2015

1. A simple method is described for the purification of the alkali and P light chains from chicken gizzard myosin. 2. The sequence of the alkali light chain has been unequivocally determined, except for the N-terminal dipeptide, by using the tryptic and CNBr peptides. 3. No evidence was obtained for any specific high-affinity Ca2+-binding sites on the alkali light chain. 4. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50120 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1983) 209, 5.
Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique
Authors: Shephard E A , Phillips I R , Bayney R M , Pike S F , Rabin B R
Published: August 2015

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.
The coenzyme-binding characteristics of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase
Authors: Hart G J , Dickinson F M
Published: August 2015

The binding of NADH and NAD+ by cytoplasmic aldehyde dehydrogenase was studied by various direct and indirect methods. At pH 7.0 at 25 degrees C there appears to be approx. 1 binding site for both nucleotides per 200 000 daltons of protein, although the NAD+-binding results are rather uncertain. Estimates of the dissociation constants of the E . NADH and E . NAD+ complexes under the stated conditions are also presented. Preparations of enzyme are sometimes found to contain significant amounts of very tightly bound NAD+ and NADH. The implications of these findings are discussed.
Arterial basement-membrane-like material isolated and characterized from rabbit aortic myomedial cells in culture
Authors: Heickendorff L , Ledet T
Published: August 2015

Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.
Antiviral activity of the 3′;-amino derivative of (E)-5-(2-bromovinyl)-2′;-deoxyuridine
Authors: De Clercq E , Descamps J , Balzarini J , Fukui T , Allaudeen H S
Published: August 2015

3′-NH2-BV-dUrd, the 3′-amino derivative of (E)-5-(2-bromovinyl)-2′-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3′-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3′-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5′-triphosphate of 3′-NH2-BV-dUrd (3′-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3′-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3′-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5′-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3′;-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.
N-alkylation of the haem moiety of cytochrome P-450 caused by substituted dihydropyridines. Preferential attack of different pyrrole nitrogen atoms after induction of various cytochrome P-450 isoenzymes
Authors: De Matteis F , Gibbs A H , Hollands C
Published: August 2015

1. 3,5-Diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) gives rise to N-ethylprotoporphyrin in the liver of rats by donating its 4-ethyl group to one of the pyrrole nitrogen atoms of haem. Four structural isomers are obtained, depending on which pyrrole nitrogen is alkylated. 2. When rats are pretreated with an inducer of cytochrome P-450, the production of N-ethylprotoporphyrin caused by 4-ethyl-DDC is greater, both in the whole animal and in hepatocytes incubated with the drug in vitro. 3. Pre-incubation of hepatocytes with 2-allyl-2-isopropylacetamide decreases the yield of N-ethylprotoporphyrin due to 4-ethyl-DDC, an effect largely reversed by adding exogenous haem. 4. The isomeric composition of N-ethylprotoporphyrin produced in vivo and in vitro depends on the cytochrome P-450 isoenzyme that predominates at the time of treatment, suggesting a role for the apo-cytochrome in directing alkylation on to one of the pyrrole nitrogens.
Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis
Authors: Hodgson J A , Lowe P N , Perham R N
Published: August 2015

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.
Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris
Authors: Green J , Haywood G W , Large P J
Published: August 2015

1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and spermidine were oxidized at both primary amino groups; in the case of spermidine this is a different specificity from that of plasma amine oxidase. 8. Under appropriate conditions, P. pastoris benzylamine/putrescine oxidase (which is very easy to purify) can be a useful analytical tool in measuring polyamines.
Kinetic parameters from progress curves of competing substrates. Application to β-lactamases
Authors: Waley S G
Published: August 2015

The use of two substrates, one of them a reporter substrate, is often convenient. The time course of an enzymic reaction with competing substrates is given explicitly in the present paper. Kinetic parameters can be readily obtained. The method is applied to the hydrolysis of benzylpenicillin, with cephalosporin C as the reporter substrate, catalysed by a Pseudomonas beta-lactamase.
Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism
Authors: Morpurgo L , Hartmann H J , Desideri A , Weser U , Rotilio G
Published: August 2015

The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).
Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase
Authors: Kasprzak A A , Papas E J , Steenkamp D J
Published: August 2015

Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated.
Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants
Authors: Hughes R C , Mills G
Published: August 2015

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer α-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.
Identification of carbohydrate-binding proteins from mouse and human fibroblasts
Authors: Roff C F , Rosevear P R , Wang J L , Barker R
Published: August 2015

Three carbohydrate-binding proteins (Mr 35 000, 16 000 and 13 500) were isolated from extracts of mouse 3T3 fibroblasts by affinity chromatography on polyacrylamide beads to which was covalently bound the ligand 6-aminohexyl 4-beta-D-galactosyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. None of these proteins bind to polyacrylamide beads coupled with either 6-aminohexanol or 6-aminohexyl β-D-galactopyranoside. Therefore they appear to be carbohydrate-binding proteins specific for galactose-terminated glycoconjugates. A carbohydrate-binding protein was also purified from extracts of human foreskin fibroblasts. This protein (Mr 35000) may represent the human counterpart of the mouse protein of similar Mr and binding properties.
Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions
Authors: Boiteux A , Markus M , Plesser T , Hess B , Malcovati M
Published: August 2015

The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis. The data were analysed on the basis of a concerted model with three conformational states [Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem. J. 189, 421-433] by using a novel procedure for a computer-directed treatment of progress curves [Markus & Plesser (1976) Biochem. Soc. Trans. 4, 361-364]. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex. We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.
The effect of counterions on melittin aggregation
Authors: Tatham A S , Hider R C , Drake A F
Published: August 2015

Melittin, a surface-active polypeptide from bee venom, has an overall hydrophobic N-terminus, with basic residues clustered at the C-terminus. In aqueous solution melittin exists as a mixture of monomer and tetramer, the monomer adopting a predominantly random-coil configuration, whereas the tetramer is rich in α-helix. The tendency of melittin to aggregate is dependent on the counter-anions present in solution, the effect being most marked with phosphate, decreasing in the order HPO4(2-) greater than SO4(2-) greater than ClO4- greater than Cl-.
Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism
Authors: Brocklehurst K , Willenbrock S J F , Salih E
Published: August 2015

The effects of selection by a small molecule, when binding to a protein, of a particular conformation from an equilibrium stereopopulation on the characteristics of the pH-dependence of reaction with a reactivity probe or substrate were determined by analysis of an appropriate kinetic model. For reaction in one protonic state containing an equilibrium mixture of two conformational isomers, the pH-second-order rate constant (k) profile is of conventional sigmoidal form. The apparent pKa value is a composite of the pKa values of the two conformational states. The value of pKapp. for a given enzyme under given experimental conditions will always be the same (provided that the site-specificity assumed in the model is maintained) irrespective of whether only one conformation reacts or both react, with the same or with different rate constants. The experimentally determined pH-independent rate constant (kapp.) is an average of the reactivities of the two conformational states weighted in favour of the predominant form. The presence of an additional but unreactive conformational state also affects the value of kapp. The possibility that overlapping acid dissociations that affect the reactivity of the enzyme might provide pH-k profiles often indistinguishable in practice from simple sigmoidal dissociation curves and subject to variability in apparent pKa values was evaluated by a simulation study. If two reactive protonic states of the enzyme respond differently to changes in the structure of the substrate or site-specific reactivity probe, differences in apparent pKa values of up to approx. 1 unit can be exhibited without deviation from sigmoidal behaviour being reliably observed. Differences in apparent pKa values observed in some site-specific reactions of papain and their possible consequences for its catalytic mechanism are discussed.
A simple procedure for purifying mammalian duodenal Ca2+-binding proteins on a 100 mg scale and an investigation of the stoichiometry of their high-affinity binding of Ca2+ ions
Authors: Bryant D T W , Andrews P
Published: August 2015

Vitamin D-dependent Ca2+-binding proteins were isolated on a 100 mg scale from the duodenal mucosae of pig, sheep and rabbit. Ion-exchange chromatography in two stages, which used a known property of the proteins, a charge difference with or without bound Ca2+ ions, was sufficient to obtain the pure proteins from several litres of heated mucosal extracts. In the preparation, treatment with a cation-exchange resin rather than with EDTA was used to obtain Ca2+-free conditions when they were required. The proteins were characterized by their amino acid compositions. All three proteins contained two tightly bound Ca2+ ions per molecule, a property now considered to be common to the mammalian Ca2+-binding proteins of this type. The labile nature of the pig Ca2+-free protein was demonstrated by the formation of a form which bound only one Ca2+ ion. Titration of the Ca2+-free binding proteins with 45Ca2+ in a flow-dialysis cell, and of Ca2+-free α-lactalbumin used as a control, demonstrated the binding stoichiometry and indicated that Ca2+ ions were bound to the mucosal proteins in dilute buffer at pH 7.5 with a Kd of the order of 10 nM.
Purification and characterization of an antibacterial protein from haemolymph of Sarcophaga peregrina (flesh-fly) larvae
Authors: Okada M , Natori S
Published: August 2015

Three antibacterial proteins were induced when the body wall of Sarcophaga peregrina (flesh-fly) larvae was injured with a hypodermic needle. These proteins were separated and one was purified to homogeneity. The molecular weight of the purified protein was 5000 and its amino acid composition was similar to that of cecropins, which are antibacterial proteins in Hyalophora cecropia (cecropia moth) pupae. This protein was found to have bactericidal activity and to be effective at a concentration of 0.1 micrograms/ml against certain Gram-negative and Gram-positive bacteria.
A qualitative and quantitative study of subfractions of the histone H10 in various mammalian tissues
Authors: Harris M R , Smith B J
Published: August 2015

The histone H10 was examined from seven mammalian species. All tissues were shown to contain two subfractions of H10, except for those of rabbit, in which little or no H10 was found. The subfraction composition was compared quantitatively in different mouse and hamster tissues, with the conclusion that this composition is tissue-specific. It is proposed that the wide occurrence of H10, together with the evidence of no more or less than two subfractions wherever it occurs, and the tissue-specific nature of the ratio of subfractions, signify that these two subfractions have specific individual functions.
Protein-mediated lipid transfer. The effects of lipid-phase transition and of charged lipids
Authors: Xü Y H , Gietzen K , Galla H J , Sackmann E
Published: August 2015

The protein-mediated phospholipid exchange between small unilamellar vesicles was investigated by fluorescence polarization measurements with diphenylhexatriene as optical probe. Thermotropic phase-transition measurements were taken after mixing two vesicle preparations of distinct and different phase-transition temperatures or having different states of charge. From the heights of each phase-transition step, we were able to follow the lipid-exchange process in the presence, as well as in the absence (natural exchange), of so-called transfer protein isolated from beef liver. A strong enhancement of the lipid transfer was observed at the corresponding lipid-phase-transition temperature, which is explained by the presence of fluctuating fluid and ordered domains co-existing at the lipid-phase-transition temperature. A unidirectional lipid transfer of the neutral component was observed between negatively charged phosphatidic acid and neutral phosphatidylcholine vesicles. Fluorescence polarization measurements showed the disappearance of the phosphatidylcholine phase transition, whereas the phosphatidic acid phase transition broadened and its phase transition temperature became lower.
Albumin binding of photobilirubin II
Authors: Meisel P , Biebler K E , Gens A , Jaehrig K
Published: August 2015

Photobilirubin II, a stereoisomer of bilirubin, binds to human serum albumin at a single binding site (K = 2.2 × 10(6)M-1), presumably the high-affinity bilirubin-binding site. Binding in the secondary (class II) binding sites is of minor importance. The results are discussed with respect to photometabolism of bilirubin and as a possible source of error in the determination of bilirubin unbound to albumin.
A proton-nuclear-magnetic-resonance study of human somatotropin (growth hormone). Assignment and properties of the histidine residues
Authors: Turner C , Cary P D , Grego B , Hearn M T W , Chapman G E
Published: August 2015

The 1H-n.m.r. spectra of human somatotropin (growth hormone) show perturbed peaks from individual aromatic and aliphatic apolar residues, characteristic of a specifically folded globular structure. The imidazole C-2-H resonances of the histidine residues (at positions 18, 21 and 151 in the somatotropin sequence) were individually resolved, and their titration behaviour in the pH range 1.2-11.5 was investigated. The imidazole C-2-H resonance of histidine-151 is assigned, by comparison of its titration behaviour in human somatotropin and desamido-somatotropin (Asn-152 leads to Asp-152). The C-2-H resonances of all three histidine residues are assigned, by comparison of their relative deuterium-exchange rates (determined by n.m.r.) and the relative tritium-exchange rates of the histidine residues (determined by tryptic digestion of tritiated human somatotropin and reversed-phase high-pressure liquid-chromatographic separation of the histidine-containing tryptic peptides). There is evidence that histidine-18 forms an ion-pair bond with a glutamic acid or aspartic acid residue. The globular structure does not appear to change from pH3 to 11.5, though there is evidence for an unfolding of a region of the structure (involving histidine-21 and a tyrosine residue) below pH3.
A glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls
Authors: Waldron K W , Brett C T
Published: August 2015

A particulate enzyme preparation made from epicotyls of 1-week-old etiolated pea (Pisum sativum) seedlings was shown to incorporate glucuronic acid from UDP-D-[U-14C]glucuronic acid into a hemicellulosic polysaccharide. Optimum conditions for the incorporation include the presence of Mn2+ ions at between 4 and 10 mmol/litre and a pH between 5 and 6. UDP-D-xylose at 1 mmol/litre allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.