bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels Diagonal peptide mapping of proteins from epidermis
Authors: Lonsdale-Eccles J D , Lynley A M , Dale B A
Published: August 2015

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.
The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies
Authors: Casu B , Oreste P , Torri G , Zoppetti G , Choay J , Lormeau J C , Petitou M , Sinaɕ P
Published: August 2015

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].
Secondary and tertiary structural differences between histone H1 molecules from calf thymus and sea-urchin (Sphaerechinus granularis) sperm
Authors: Giancotti V , Russo E , Cosimi S , Cary P D , Crane-Robinson C
Published: August 2015

Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.
Characterization of potato (Solanum tuberosum) lactate dehydrogenase isoenzymes by affinity chromatography and hybridization
Authors: Jervis L.
Published: August 2015

The five isoenzymes of potato (Solanum tuberasum) lactate dehydrogenase have been resolved by affinity chromatography. Mixtures of isoenzymes LDH-1 and LDH-5 dissociate and reassociate during freezing and thawing to produce five isoenzymes. These results indicate that potato lactate dehydrogenase isoenzymes are primary isoenzymes of the vertebrate type, which are composed of two subunit types.
Activity of phosphatidylcholine-transfer protein from spinach (Spinacia oleracea) leaves with mitochondria and chloroplasts
Authors: Julienne Marianne , Vergnolle Chantal , Kader Jean-Claude
Published: August 2015

A low-molecular-weight protein catalysing the transfer of phosphatidylcholine from liposomes to mitochondria and chloroplasts has been isolated from spinach (Spinacia oleracea) by chromatography on Sephadex G-75.
Identification of aspirinase with one of the carboxylesterases requiring a thiol group
Authors: White K N , Hope D B
Published: August 2015

Aspirin-hydrolysing activity in guinea-pig liver is located mainly in the microsomal fraction. This activity was found by electrophoresis to be due to a single carboxylesterase band, out of 12 bands revealed with alpha-naphthyl acetate as substrate. The activity is inhibited completely and irreversibly by the carboxylesterase inhibitor bis-(-4-nitrophenyl) hydrogen phosphate, and also by thiol-blocking reagents.
Essential charged amino acids in the binding of fibronectin to gelatin
Authors: Vuento M , Salonen E , Osterlund K , Stenman U H
Published: August 2015

The binding of fibronectin to gelatin-agarose was strictly dependent on pH, having a pH optimum of 7-9. The binding was strongly inhibited by increasing ionic strength. A chemical modification of lysyl and arginyl groups of fibronectin abolished the binding activity. The anionic detergents sodium dodecyl sulphate and sodium deoxycholate in concentrations of 10-100mM had the same effect. The binding was not affected by the non-ionic detergents Triton X-100, Tween 20 or Lubrol WX. The results demonstrate an important role of ionic interactions in the binding of fibronectin to gelatin. Absence of inhibition by non-ionic detergents suggests that hydrophobic interactions contribute relatively little to the binding of fibronectin to gelatin.
Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase
Authors: Pettigrew G W , Seilman S
Published: August 2015

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.
The catalytic chain of human complement subcomponent C1. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments
Authors: Arlaud G J , Gagnon J , Porter R R
Published: August 2015

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the ‘charge-relay’ system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the ‘histidine loop’, a disulphide bond that is present in all other known serine proteinases.
Presence of an a typical thermolabile species of β-hexosaminidase B in metastatic-tumour tissue of human liver
Authors: Alhadeff J A , Holzinger R T
Published: August 2015

An atypical thermolabile species of Hex B (hexosaminidase B) has been found in metastatic-tumour sites of human liver which has a thermostability curve similar to that of Hex A (hexosaminidase A), which is present in decreased amounts relative to the Hex A isoenzyme, and which exhibits decreased relative activity at acidic pH values (2.6-3.6) when compared with control-liver Hex B. This atypical Hex B isoenzyme has a normal apparent Michaelis constant (0.6 mM) for 4-methylumbelliferyl 2-deoxy-2-acetamido-beta-D-glucopyranoside. The presence of this atypical Hex B suggests that variant beta-chains are being produced in metastatic-tumour tissue.
Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate
Authors: Brocklehurst K , Mushiri S M , Patel G , Willenbrock F
Published: August 2015

1. The pH-dependences of the second-order rate constants (k) for the alkylation by chloroacetate of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) were determined over a wide range of pH at 25 degrees C at I 0.1. 2. The main feature of both pH-k profiles is a striking rate maximum at pH6 (characterizing parameters in both cases pKI approx. 3.5, pKII approx. 8.4 and pH-independent rate constant approximately kXH 2.5-3.0 M-1 . s-1). 3. The profile for the ficin reaction contains a plateau at high pH, with approximately kX 0.10 M-1 . s-1; if an analogous plateau exists in the papain reaction, approximately kX ix much lower, less than 0.02 M-1 . s-1. 4. Both enzymes appear to contain closely similar thiolate-imidazolium interactive systems at pH6, but differences in their behaviour in more-acidic media and in alkaline media suggest differences in interaction with the postulated carboxylate component of the putative catalytic triad.
Fast-atom-bombardment mass spectrometry. A new technique for the determination of molecular weights and amino acid sequences of peptides
Authors: Williams D H , Bradley C V , Santikarn S , Bojesen G
Published: August 2015

A detailed study of the mass spectra of peptides produced by the new technique of fast-atom bombardment is reported. Molecular weights of unmodified peptides containing up to 21 amino acids have been determined. In favourable cases, the molecular-weight determination may be made on as little as 0.1 nmol of sample. Positive-ion and negative-ion spectra are obtained with equal facility. With sample sizes in the range 2-50nmol, sequence information can be obtained in many cases. The technique represents an important contribution to structural studies on peptides, since (i) blocked peptides may be studied, (ii) no prior formation of chemical derivatives is necessary (except for distinction between lysine and glutamine), (iii) sensitivity is good, (iv) large peptides, and, in some cases, mixtures of peptides, can be studied, and (v) the technique is easy to operate, with ions being produced over a long period (5-30 min).
Purification and properties of a proteolytic enzyme from the cercariae of the human trematode parasite Schistosoma mansoni
Authors: Landsperger W J , Stirewalt M A , Dresden M H
Published: August 2015

Skin penetration by the cercarial stage of the human trematode parasite Schistosoma mansoni is mediated by the secretion of proteolytic enzymes able to digest components of mammalian connective tissues. In the present study the purification of these proteinases from cercarial homogenates is reported. The major proteinase species has a mol. wt. of approx. 25 000 and exists in monomeric form as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This proteinase has an isoelectric point of 6.0. Studies presented here, with a variety of substrates and inhibitors, confirm previous claims that these proteinases belong to the serine class, and, in addition, suggest that they resemble the vertebrate chymotrypsins rather than trypsins or elastases. However, the amino acid composition of the cercarial proteinase differs significantly from bovine chymotrypsin and from the human leucocyte chymotrypsin-like cathepsin G. The amino-acid-composition differences between these proteinases are consistent with their differences in isoelectric point. In order to obtain an insight into the role of the proteinase in skin penetration, its activity on cartilage proteoglycan monomers and on the isolated peptide backbone of proteoglycan was studied. The results of the present study indicate that the cercarial enzyme catalyses a limited specific digestion of the peptide core.
Mechanism of inhibition of Escherichia coli RNA polymerase by captan
Authors: Dillwith J W , Lewis R A
Published: August 2015

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.
Concerning the structure of photobilirubin II
Authors: Stoll M S , Vicker N , Gray C H , Bonnett R
Published: August 2015

Evidence is presented which supports the postulate that the photobilirubins IIA and IIB are diastereoisomers in which the C-3 vinyl group has cyclized intramolecularly. The evidence comes principally from proton n.m.r. spectroscopy at 400 MHz and from chemical considerations. The cyclic structures require the E-configuration at the C-4 double bond in the precursor; this is the first structural evidence for the Z leads to E isomerization in bilirubin and supports the view that the precursor (photobilirubin IA or IB) is (4E, 15Z)-bilirubin. Brief irradiation of photobilirubin II gives bilirubin, a new compound (photobilirubin III) and unchanged starting material. The various photoisomers are discussed in terms of their inter-relationships and biological fates.
L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L
Authors: Barrett A J , Kembhavi A A , Brown M A , Kirschke H , Knight C G , Tamai M , Hanada K
Published: August 2015

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine
Authors: Pelosi P , Baldaccini N E , Pisanelli A M
Published: August 2015

2-Isobutyl-3-methoxypyrazine, a potent bell-pepper odorant, binds to cow olfactory mucosa homogenate. The receptor is saturable in the micromolar range and is competitively inhibited by other bell-pepper odourants, but not by other pyrazines of different odours. Other tissues do not bind 2-isobutyl-3-methoxypyrazine at a significant extent. We suggest that this receptor is involved in odour discrimination.
A cytochrome c methyltransferase from Crithidia oncopelti.
Authors: Valentine J , Pettigrew G W
Published: August 2015

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.
Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.
Authors: Heimer Y M , Mizrahi Y
Published: August 2015

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 × 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.
Glycoprotein biosynthesis in quiescent and stimulated thymocytes and a T-cell lymphoma. The synthesis of high-mannose oligosaccharides
Authors: Rupar C A , Cook G M
Published: August 2015

Quiescent thymocytes, mitogen-stimulated thymocytes and acute-leukaemic lymphoblasts provide a model for the study of protein glycosylation in quiescent cells, mitotically active non-malignant and malignant cells respectively. The biosynthesis of both complex and high-mannose-type oligosaccharides was monitored by metabolic labelling with [6-3]fucose and [2-3H]mannose. Bio-Gel P6 elution profiles of [6-3H]fucose-labelled glycopeptides showed that quiescent thymocytes and stimulated thymocytes synthesized qualitatively and quantitatively similar glycopeptides; however, higher-molecular-weight glycopeptides were synthesized by the acute-leukaemic lymphoblasts. The amount of [2(-3)H]mannose incorporated into glycopeptide by quiescent thymocytes was less than 10% of that incorporated by stimulated thymocytes. The Bio-Gel P6 elution profile of [2(-3)H]mannose-labelled glycopeptides from acute leukaemic lymphoblasts was qualitatively similar to that of stimulated thymocytes, with about 40% of the radioactivity incorporated into one glycopeptide peak. This glycopeptide was characterized by Bio-Gel P6 and concanavalin A affinity chromatography, radioactive-sugar analysis, sensitivity to alpha-mannosidase and endoglycosidase H and resistance to beta-glucosaminidase as containing a high-mannose oligosaccharide, possible of Man7-8GlcNAc2 structure. Pulse/chase experiments indicated that this high-mannose oligosaccharide was an end product and not a biosynthetic intermediate. It is concluded that higher-molecular-weight fucose-labelled glycopeptides are characteristic of the malignant cell type, and the synthesis of high-mannose oligosaccharide, Man7-8GlcNAc2, in stimulated thymocytes and acute-leukaemic lymphoblasts is associated with mitotically active cells.
Evolutionary significance of the renal excretion of transferrin half-molecule fragments
Authors: Williams John , Grace Sheila A. , Williams Jennifer M.
Published: August 2015

It is generally thought that the duplicated structure of serum transferrin in vertebrates arose by gene duplication and fusion from a small ancestral protein. We have found that the isolated domains of transferrin are rapidly lost from the bloodstream via the kidneys. Therefore we suggest that the ancestral transferrin was not a serum protein or, alternatively, that it was not as small as the half-molecule.
Biosynthesis of dermatan sulphate. Assay and properties of the uronosyl C-5 epimerase
Authors: Malmström A , Aberg L
Published: August 2015

During biosynthesis of dermatan sulphate D-glucuronate (GlcA) residues are converted to L-iduronate (IdoA) residues via the reaction [Formula: see text]. The reaction occurs on the polymer level and is catalysed by a C-5 uronosyl epimerase. The reversible release of the C-5 hydrogen was utilized as a measure of the enzyme activity with 5-3H-labelled chondroitin as a substrate. 3H released during incubation was distilled and quantified by liquid-scintillation counting. The epimerase has a low pH optimum (5.6) and requires divalent cations, Mn2+ being the most efficient for activity. The Km for chondroitin is 1.2 × 10(-4) M. The epimerase is largely associated with the microsomal fractions (90%). Two-thirds of the activity can be solubilized by detergents. Microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for the biosynthesis of heparan sulphate and one for that of dermatan sulphate. The two epimerases have different cofactor and pH requirements.
The effect of salt concentration on the iron-binding properties of human transferrin
Authors: Williams J , Chasteen N D , Moreton K
Published: August 2015

The salt dependence of the iron-binding properties of transferrin was studied by urea/polyacrylamide-gel electrophoresis. The distribution of iron between the N-terminal and C-terminal binding sites under equilibrium conditions and the rates of release of iron from the two sites were studied. It was found that salt increases the thermodynamic stability of iron binding in the N-terminal site relative to the C-terminal site. Similar behaviour is observed for the kinetics of iron release, where salt retards the rate of removal of iron from the N-terminal site but facilitates removal from the C-terminal site.
Isolation, characterization and N-terminal sequences of the CNBr-cleavage peptides from human complement Factor B. Localization of a free thiol group and a sequence defining the site cleaved by factor D
Authors: Christie D L , Gagnon J
Published: August 2015

Nine CNBr-cleavage peptides from Factor B (a component of the alternative pathway of complement) were isolated. Each was characterized by amino acid analysis and automated Edman degradation. One peptide contained a methionyl bond resistant to cleavage by CNBr. The number of CNBr-cleavage peptides is in agreement with the results of amino acid analysis of Factor B and the fragments Ba and Bb. A total of 358 unique residues were identified from the N-terminal sequences of the CNBr-cleavage peptides. These represent approx. 50% and 60% of the total residues of Factor B and fragment Bb respectively. Alignment of two CNBr-cleavage peptides (CB-VIc and CB-IV) provided a continuous segment of 140 residues. This sequence contained the site cleaved by Factor D to generate the Ba and Bb fragments during the activation of complement. Peptide CB-IV contained a free thiol group at a position corresponding to residue 33 of fragment Bb. Amino sugar analyses of Factor B and of fragments Bb and Ba indicated that all the carbohydrate structures of factor B are N-linked to asparagine through N-acetylglucosamine. The two carbohydrate-attachment sites of the Bb fragment were identified.
Active sites of β-lactamases. The chromosomal β-lactamases of Pseudomonas aeruginosa and Escherichia coli
Authors: Knott-Hunziker V , Petursson S , Jayatilake G S , Waley S G , Jaurin B , Grundström T
Published: August 2015

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.
The stereochemical course of phosphoryl transfer catalysed by glucose 6-phosphatase
Authors: Lowe G , Potter B V L
Published: August 2015

Rat liver microsomal glucose 6-phosphatase catalyses phosphoryl transfer between D-glucose 6-[(R)-16O,17O,18O]phosphate and D-glucose with retention of configuration at the phosphorus atom. Since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with a phosphoryl-enzyme intermediate. Such an intermediate has been proposed previously from kinetic and 32P-labelling experiments.
Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites
Authors: Atassi M Z , Sakata S
Published: August 2015

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.
Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines
Authors: Fekkes D , Hennemann G , Visser T J
Published: August 2015

The enzymic 5′-deiodination of 3′,5′-di-iodothyronine and 5-deiodination of 3,3′,5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3′,5′-Di-iodothyronine was a competitive inhibitor of 3,3′,5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3′,5-tri-iodothyronine was a competitive inhibitor of 3′,5′-di-iodothyronine 5′-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.
The N-terminal ubiquitin-binding region of ubiquitin-specific protease 28 modulates its deubiquitination function: NMR structural and mechanistic insights
Authors: Wen Yi , Shi Li , Ding Yiluan , Cui Rong , He Wen-tian , Hu Hong-yu , Zhang Naixia
Published: August 2015

The deubiquitinase ubiquitin-specific protease 28 (Usp28) contains a ubiquitin-binding region (UBR) composed of one ubiquitin-associated domain (UBA) and one ubiquitin-interacting motif (UIM) at its N-terminus. It is of interest that an additional small ubiquitin-like modifier (SUMO)-interacting motif (SIM) is located next to its UIM. To date, the functional role of the Usp28 UBR is still not understood. To elucidate the regulatory mechanism of the UBR on the full functional display of Usp28, in the present study, NMR and biochemical approaches were applied. The solution structure of Usp28 UBR was obtained, and the key residues responsible for ubiquitin and SUMO1/2 recognition were identified. In addition, we find that the ubiquitin-binding ability of Usp28 UBR was required for full enzymatic activity of Usp28, whereas binding of SUMO1/2 impaired the catalytic activity of the enzyme by competitively blocking its interactions with ubiquitin substrates. Our findings provide a first insight into understanding how the enzymatic activity of Usp28 is regulated by its non-catalytic UBR and endogenous ligands.
A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions
Authors: Orlando Kelly A. , Iosue Christine L. , Leone Sarah G. , Davies Danielle L. , Wykoff Dennis D.
Published: August 2015

Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2.
Isolation and characterization of human cervical-mucus glycoproteins
Authors: Carlstedt I , Lindgren H , Sheehan J K , Ulmsten U , Wingerup L
Published: August 2015

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).
Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum
Authors: Anastasi A , Brown M A , Kembhavi A A , Nicklin M J H , Sayers C A , Sunter D C , Barrett A J
Published: August 2015

The protein from chicken egg white that inhibits cysteine proteinases, and has been named ‘cystatin’, was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 × 10(-11)M), cathepsin B (8 × 10(-10)M), cathepsin H (about 2 × 10(-8)M) and cathepsin L (about 3 × 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 × 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.
Amelogenins. Sequence homologies in enamel-matrix proteins from three mammalian species
Authors: Fincham A G , Belcourt A B , Termine J D , Butler W T , Cothran W C
Published: August 2015

Partial amino acid sequences for selected amelogenin polypeptides isolated from the developing enamel of cow, pig and human foetuses are reported. It was found that there was an identity of sequence for the initial 28 residues of the polypeptides analysed, irrespective of their origin or size. A tyrosine-rich polypeptide was shown to be the N-terminal fragment of the principal higher-molecular-weight amelogenins, although a leucine-rich polypeptide of similar size was not identified in any other amelogenin structure. The findings demonstrate a striking degree of sequence conservation for the amelogenin proteins of the extracellular enamel matrix and support the concept of a discrete fragmentation of an initial 30 000 Da amelogenin molecule during the mineralization of the enamel.
A new isotype sequence (V κ 27) of the variable region of κ-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides
Authors: Chang J Y , Herbst H , Aebersold R , Braun D G
Published: August 2015

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.
Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase
Authors: Prehm P
Published: August 2015

Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.
Equilibria amongst different molybdenum (V)-containing species from sulphite oxidase. Evidence for a halide ligand of molybdenum in the low-pH species
Authors: Bray R C , Gutteridge S , Lamy M T , Wilkinson T
Published: August 2015

The interaction of chloride, fluoride and phosphate ions with the molybdenum centre of sulphite oxidase in the pH range 6.2 to 9.6 has been studied by e.p.r. of Mo(V) in the enzyme reduced by sulphite. Detailed studies were made from e.p.r. spectra recorded at about 120K and more limited studies from spectra of liquid samples at about 295K and also from enzyme activity measurements. Interconversion between low-pH and high-pH Mo(V) e.p.r. signal-giving species [described by Lamy, Gutteridge & Bray (1980) Biochem. J. 185, 397-403] is influenced by chloride concentration, a 10-fold increase in concentration (in the range of about 1 mM to 100 mM) causing an increase of about 1 pH unit in the apparent pK for the conversion. This suggests that chloride is a constituent of the low-pH species. In support of this, high concentrations of fluoride modified the e.p.r. spectrum. Partial conversion to a Mo(V) species, in which F- has presumably replaced Cl- and showing hyperfine coupling of A(19F)av. 0.5mT, is indicated. It is proposed that interconversion between high-pH and low-pH species is of the form: (formula; see text) No evidence that Cl- is essential for enzymic activity was found. Data relating to equilibria amongst low-pH, high-pH and also the phosphate species are presented. Depending on pH and on concentrations of Cl- and H2PO4-, one, two, or all three species may be present. Qualitatively, under appropriate conditions, the phosphate species tends to replace some or all of the low-pH species. Quantitative analysis by a computer procedure permitted an appropriate scheme to be deduced and equilibrium constants to be evaluated. Studies on the e.p.r. signals at 295K indicated that similar equilibria applied in liquid solution, but with some changes in the values of the constants. The structure of the molybdenum centre in its various states and the nature of the enzymic reaction are discussed.
The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.
Authors: Irvine G B , Blumsom N L , Elmore D T
Published: August 2015

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.
Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta
Authors: Odermatt E , Risteli J , van Delden V , Timpl R
Published: August 2015

Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.
Electron-microscopical approach to a structural model of intima collagen
Authors: Furthmayr H , Wiedemann H , Timpl R , Odermatt E , Engel J
Published: August 2015

Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.