bluuebunny/crossref_metadata_2025 huggingface dataset:
The presence of a cartilage-like proteoglycan in the adult human meniscus
Authors: Roughley P J , McNicol D , Santer V , Buckwalter J
Published: August 2015

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This ‘cartilage-like’ proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the ‘cartilage-like’ proteoglycan, which does not contain dermatan sulphate.
Inhibition of rabbit tissue kininase by anti-(endo-oligopeptidase A) antibodies
Authors: Coelho H L L , Cicilini M A , Carvalho K M , Carvalho I F , Camargo A C M
Published: August 2015

Highly purified rabbit brain endo-oligopeptidase A injected into goats produced, after 60 days of immunization, antisera that specifically inhibit purified rabbit brain endo-oligopeptidase A. An immunoreactive kininase having the same specificity as rabbit brain endo-oligopeptidase A for bradykinin was detected in several rabbit tissues. The highest amount of this immunoreactive kininase was found in the 25000 g supernatant fraction (S fraction) of heart, liver, skeletal muscle, ovary, brain and testis homogenates, corresponding to 89, 86, 78, 59, 56 and 53% respectively of the whole kininase activity found in the S fraction.
Detection of ligand-induced perturbations affecting the biotinyl group of mammalian acetyl-coenzyme A carboxylase by using biotin-binding antibodies
Authors: Ahmad Fazal , Ahmad Patricia M. , Dickstein Ross , Greenfield Ed
Published: August 2015

Biotin-binding antibodies were raised in rabbits by injecting biotin–bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1μm), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody–carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer–antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg2+, ATP+Mg2+ or ATP+Mg2++HCO3−, however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s∼30–45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a ∼19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.
The primary structure of the constant region of Basilea-rabbit immunoglobulin λ-chains
Authors: Garcia I , Jaton J C
Published: August 2015

The amino acid sequence of the constant region of the immunoglobulin lambda-chain of the rabbit (C lambda) has been determined. This chain was obtained from a homogeneous anti-(type II pneumococcal polysaccharide) antibody fraction raised in Basilea rabbit 4548. The C lambda-region sequence was established by the analysis of tryptic and some overlapping chymotryptic peptides and partly by homology with known C lambda-region sequences of lambda-chains from other species. Peptides were isolated by a combination of methods including high-voltage paper electrophoresis, gel filtration and high-pressure liquid chromatography. The peptides were subjected to automated Edman degradation in the presence of Polybrene. The sequence showed no evidence of heterogeneity. Large interspecies similarities, as well as amino acid interchanges, are apparent among various C lambda regions; the degrees of homology between rabbit C lambda region and the C lambda region from human, porcine and murine chains are 72.6, 71 and 72% respectively. The similarity of lambda-chains of different species is much greater than that found between rabbit kappa- and lambda-chains (about 34% homology). Genetic implications of these results are discussed.
Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans Isolation, fractionation and chemical characterization
Authors: Kapoor R , Phelps C F , Cöster L , Fransson L A
Published: August 2015

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.
The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli
Authors: Graham A
Published: August 2015

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.
The influence of pressure on the self-assembly of the thick filament from the myosin of vertebrate skeletal muscle
Authors: Davis J S
Published: August 2015

The thick-filament-monomeric-myosin equilibrium was prepared from pure myosin at pH 8.1. The application of hydrostatic pressure to the self-assembly equilibrium resulted in a biphasic dissociation curve in which a linear decrease in turbidity (a measure of weight added to or lost from the filament) was followed by a transition to a second pressure-insensitive phase. The first phase represents the effect of hydrostatic pressure on the growth or propagation phase of filament assembly. Here is was shown that hydrostatic pressure served to shorten the filaments in concert towards the bare zone whilst maintaining the narrow length distribution seen at atmospheric pressure; the filament concentration remained constant during the experiment. A more precise definition of the delta-v for the assembly of monomer into filament was obtained than had hitherto been possible. The positioning of the bare zone at the centre of the filament seems to be one of the more obvious functions of the length-regulation mechanism. It also appears that all the basic structural elements of the native thick filament are potentially present in the pH 8.1 homopolymer; its length can be increased by physiological concentrations of MgCl2 and decreased by pressure. The monodisperse native filament could then be formed by a fine tuning of the basic length-regulation mechanism of the homopolymer by the co-polymerization of the other thick-filament proteins.
Pressure-jump studies on the length-regulation kinetics of the self-assembly of myosin from vertebrate skeletal muscle into thick filament
Authors: Davis J S
Published: August 2015

The self-assembly of myosin monomer into thick filament occurs via a two-step mechanism. At first a pair of myosin monomers reacts to form a parallel dimer; the dimer in turn adds to the filament ends at a rate that is independent of filament length. The rate of the dissociation reaction on the other hand is length-dependent. The ‘off’ rate constant has been shown to increase exponentially by a factor of 500 as the filament grows from the bare-zone out to its full length. The length of the filament is thus kinetically controlled; myosin is added to the filament at a fixed rate, whereas the dissociation rate increases to a point where equilibrium is established and the filament ceases to grow. The structural implications implicit in the mechanism are discussed.
Cell-free biosynthesis of erythroglycan in a microsomal fraction from K-562 cells
Authors: Russin T Z , Laine R A , Turco S J
Published: August 2015

Particulate membrane preparations from K-562 [human CML (chronic-myelogenous-leukaemia)-derived] cells catalyse the transfer of [3H]galactose from UDP-[3H]-galactose and [3H]N-acetylglucosamine from UDP-[3H]N-acetylglucosamine into an endogenous product that on digestion with Pronase yields long-chain glycopeptides (mol.wt. 7000-10 000) called ‘erythroglycan’. Incorporation of either labelled sugar increased up to 60 min of incubation time. The labelled erythroglycan was isolated by chromatography on Sephadex G-50 and characterized by digestion with endo-beta-galactosidase from Escherichia freundii, followed by analysis on Bio-Gel P-2 and paper chromatography. This digestion gave the following four products: (1) a disaccharide with the sequence beta GlcNAc-beta Gal; (2) a trisaccharide with the sequence betaGal-betaGlcNAc-beta Gal; (3) a larger oligosaccharide containing galactose and N-acetylglucosamine; and (4) a putative protein-linkage region.
Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate
Authors: Griffin D C , Landon M
Published: August 2015

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.
Characterization of N-glycosylated type I collagen in streptozotocin-induced diabetes
Authors: Le Pape A , Muh J P , Bailey A J
Published: August 2015

The N epsilon-glycosylation of lysine and hydroxylysine residues in collagen from streptozotocin-induced-diabetic rats was confirmed and the stability of the complex shown to be due to an Amadori rearrangement. The studies also demonstrate the relative specificities of glucose, galactose and mannose in their reaction with collagen. The glycosylation of lysine in vitro occurs with glucose and galactose, but not with mannose, whereas only gucose reacts with hydroxylysine to any significant extent. Glycosylation of collagen occurs slowly during normal aging, but in contrast with reports suggesting accelerated aging of collagen in diabetic animals, we clearly demonstrated that the apparent increased stability is not due to an acceleration of the normal maturation process involving the reducible cross-links.
Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii
Authors: Leadlay P F
Published: August 2015

Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+.
The purification and molecular properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa
Authors: Nimmo G A , Coggins J R
Published: August 2015

Neurospora crassa contains three isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, which are inhibited by tyrosine, tryptophan and phenylalanine respectively, and it was estimated that the relative proportions of the total activity were 54%, 14% and 32% respectively. The tryptophan-sensitive isoenzyme was purified to homogeneity as judged by polyacrylamide-gel electrophoresis and ultracentrifugation. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes were only partially purified. The three isoenzymes were completely separated from each other, however, and can be distinguished by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34 and polyacrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the tryptophan-sensitive isoenzyme contained one type of subunit of molecular weight 52000. The molecular weight of the native enzyme was found to be 200000 by sedimentation-equilibrium centrifugation, indicating that the enzyme is a tetramer, and the results of cross-linking and gel-filtration studies were in agreement with this conclusion.
Borate inhibits activation of inactive dinitrogenase reductase from Rhodospirillum rubrum
Authors: Ludden Paul W.
Published: August 2015

Borate and aminophenylboronic acid were tested as inhibitors of activation of inactive dinitrogenase reductase from Rhodospirillum rubrum. Inhibition was specific for activation because activity of the active form of the enzyme was not inhibited. Inhibition showed the pH-dependence expected if borate inhibits by binding to cis-hydroxy groups of the modifying group found on the inactive enzyme.
Partial characterization of the carbohydrate units of rat intestinal sucrase-isomaltase
Authors: Herscovics A , Quaroni A , Bugge B , Kirsch K
Published: August 2015

Sucrase-isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.
Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield
Authors: Afting E G , Recker M L
Published: August 2015

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.
Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines
Authors: Wightman Paul D. , Dahlgren Mary Ellen , Hall James C. , Davies Philip , Bonney Robert J.
Published: August 2015

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.
Isolation and characterization of rat plasma fibronectin
Authors: Weiss R E , Reddi A H
Published: August 2015

Rat plasma fibronectin has been isolated and characterized and monospecific antibodies were prepared to it. Two components of fresh rat plasma (in the presence of proteinase inhibitors) bound to a gelatin-Sepharose affinity column. One protein was eluted with 4.0 M-urea and was identified as fibronectin. Another protein was eluted from the gelatin-Sepharose column with 8.0 M-urea and was identified as a 70 000-Mr collagen-binding molecule. This 70 000-Mr fragment was found to be a normal constituent of blood plasma, and its presence did not represent a proteolytic degradation product formed during isolation. The antibodies prepared against rat fibronectin only weakly cross-reacted with plasma fibronectins of chicken, horse and human. These studies shed light on the metabolic interrelationships between fibronectin and other collagen-binding molecules.
Primary structure of the ovine pituitary follitropin β-subunit
Authors: Sairam M R , Seidah N G , Chrétien M
Published: August 2015

The complete amino acids sequence of the ovine pituitary follitropin beta-subunit was established by studying the tryptic, chymotryptic and thermolytic peptides. The N-terminal sequence of the subunit was confirmed by subjecting the oxidated protein to Edman degradation in an automated sequenator. Automated Edman degradation of the reduced and alkylated (with iodo [14C]acetamide) beta-subunit indicated that most of the molecules used in the sequence studies had lost the N-terminal serine residue. This also confirmed the location of the first five half-cystine residues in the sequence. The proposed structure shows the presence of 111 amino acid residues with the two oligosaccharide moieties linked to asparagine residues located at positions 6 and 23. Heterogeneity occurs at both the termini of the polypeptide chain. Comparison of the sequence of beta-subunit of the ovine hormone with that proposed for human follitropin beta-subunit shows the absence of any deletions in the middle of the peptide chain. Of the 13 replacements, 11 residues can be explained on the basis of a single base change in the codon. The single tryptophan residue of the follitropin occupies an identical position in all the four species that have been studied. The region corresponding to residues 63-105 of the ovine beta-subunit is highly conserved in all the species.
Evidence for two forms of reverse transcriptase in human placenta of a patient with breast cancer Purification and biochemical characterization of the enzymes
Authors: Vogel A , Chandra P
Published: August 2015

Two DNA polymerases with properties of viral RNA-directed DNA polymerase were found in the placenta of a patient with breast cancer. Both enzyme activities were purified by column-chromatographic procedures or by preparative isoelectric focusing. The most distinguishing feature of the two enzymes is their specificity to transcribe (rA)n . (dT)12 or (rC)n . (dG)18. The two enzymes differ with respect to their elution profiles from the phosphocellulose column, isoelectric point, molecular weight, bivalent-cation requirements and thermal stability. Serological analysis of the (rA)n . (dT)12-activated enzyme showed that this enzyme is immunologically not related to DNA polymerase-gamma, or to any of the reverse transcriptases purified from retroviruses of avian, murine and subprimate origin. However, the activity of this enzyme was neutralized by antibodies to reverse transcriptase purified from human spleen of a patient with myelofibrosis [Chandra & Steel (1977) Biochem. J. 167, 513-524]. Attempts to purify reverse transcriptase of normal human placenta were repeatedly unsuccessful. Once the crude homogenate of normal placenta was freed from endogenous nucleic acids, no (rC)n. (dG)18-dependent activity cold be detected. U
Interaction of coenzyme M and formaldehyde in methanogenesis
Authors: Romesser J A , Wolfe R S
Published: August 2015

Chemical reaction of coenzyme M, sodium 2-mercaptoethanesulphonate (HS-CoM, Na+), and formaldehyde formed sodium 2-(hydroxymethylthio)ethanesulphonate (HOCH2-S-CoM), whereas reaction with the ammonium salt of HS-CoM yielded iminobis-[2-(methylthio)ethanesulphonate], monoammonium salt [NH = (CH2 - S - CoM)2]. In water, NH = (CH2 - S - CoM)2 decomposed to 2-(aminomethylthio)ethanesulphonate (NH2CH2 - S - CoM) and HOCH2-S-CoM. NH-2-CH2 - CoM was degraded further to form more HOCH2-S-CoM. The structures of these coenzyme M derivatives were confirmed by i.r. and n.m.r. spectroscopy and by elemental analysis. When added to cell extracts of Methanobacterium thermoautotrophicum, methane was formed from either HOCH2 - S - CoM or NH = (CH2 - S - CoM)2 at rates comparable with the rate of methane formation from the methanogenic precursor 2-(methylthio)-ethanesulphonate (CH3 - S - CoM). Formaldehyde was reduced to methane at similar rates. In addition, certain hemimercaptals, including thiazolidine and thiazolidine-4-carboxylate, were reduced, although at slower rates. The reduction of formaldehyde, thiazolidine, or thiazolidine-4-carboxylate required catalytic amounts of HS-CoM. ATP was required by cells extracts for reduction of each of these methane precursors.
Subunit structured of pig small-intestinal brush-border aminopeptidase N
Authors: Benajiba A , Maroux S
Published: August 2015

Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.
Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin
Authors: Suenson Elisabeth , Thorsen Sixtus
Published: August 2015

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α2-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α2-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Lys76, Glu1–Arg67or Glu1–Lys77)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α2-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α2-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Val441) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α2-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α2-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.
Isolation and partial characterization of two antigenic glycoproteins from rye-grass (Lolium perenne) pollen
Authors: Howlett B J , Clarke A E
Published: August 2015

Two glycoproteins have been purified from a buffer extract of rye-grass (Lolium perenne) pollen. Both migrated as single bands on sodium dodecyl sulphate/polyacrylamide gels. Glycoprotein 1 (0.8 mg/g of pollen) had a apparent mol.wt. of 33 000 and contained 95% protein and 5% carbohydrate. The monosaccharides glucose, galactose, mannose, arabinose and N-acetylglucosamine were present in the proportions 3:3:1:2:1. Glycoprotein 2 (0.4 mg/g of pollen) had an apparent mol. wt. of 68000 and contained 88% protein and 12% carbohydrate. The monosaccharides glucose, galactose, mannose, fucose, xylose, arabinose and N-acetylglucosamine were present in the proportions 4:7:13:5:8:6:6. This glycoprotein bound concanavalin A and Lotus tetragonolobus (asparagus pea) lectin. Radioallergosorbent (RAST) inhibition tests showed that Glycoprotein 1 is an effective allergen, whereas Glycoprotein 2 has less allergenic activity. A method for performing both lectin-binding assays and RAST inhibition tests using microtitre trays is described.
Subcellular localization of aldehyde reductase activities in ox brain
Authors: Ryle C M , Tipton K F
Published: August 2015

The distribution of the two principal isoenzymes of aldehyde reductase (EC 1.1.1.2) has been studied in ox brain. The more active of these, which has been termed the high-Km enzyme, has been shown to be located in the cytosol and the less abundant low-Km form has a similar localization. p-Nitrobenzaldehyde, which has been used as a substrate in previous studies, caused the reduction of NADH in the presence of the mitochondrial fraction, but mixed substrate experiments with 1,3-dinitrobenzene and the effects of pH on the activity indicate that this is due to the presence of a nitro reductase activity which has been recently described (Köchli, Wermuth & von Wartburg (1980) Biochim. Biophys. Acta 616, 133-142] rather than to the low-Km aldehyde reductase activity. Fractionation of the mitochondria indicated this activity to be largely confined to the mitochondrial inner membrane.
The effect of inorganic phosphate on the stability of some enzymes
Authors: Rippa M , Signorini M , Bellini T
Published: August 2015

In the presence of 5nM-P1, 6-phosphogluconate dehydrogenase from the yeast Candida utilis is more resistant to proteolysis and to the inactivating action of some chemical and physical agents. P1 also protects other enzymes against proteolysis. A hypothesis for the mechanism of the stabilizing action of P1 is advanced.
Collagen cross-linking compounds in human urine
Authors: Gunja-Smith Zeenat , Boucek Robert J.
Published: August 2015

We report the isolation and chemical characterization of collagen cross-linking compounds, 3-hydroxypyridinium and dihydroxylysinonorleucine, from human urine.
Isolation and partial characterization of pancreatic polypeptide-like material in the brain of the blowfly Calliphora vomitoria
Authors: Duve H , Thorpe A , Neville R , Lazarus N R
Published: August 2015

Using 10(6) flies (5 kg of heads) a pancreatic polypeptide-like material has been partially purified from the blowfly Calliphora vomitoria. The isolated material was eluted on Sephadex G-50 similarly to bovine pancreatic polypeptide and had an RF on polyacrylamide-gel electrophoresis that was identical with that of the bovine hormone. The material diluted linearly and showed parallelism with bovine standards in a bovine pancreatic polypeptide immunoassay. In specificity controls the immunoreactivity was not abolished by trasylol and no cross-reactivity was discerned in assay for glucagon, proangiotensin and cyclic AMP. These data suggest that the pancreatic polypeptide material in the brain of the blowfly has close structural similarity to the mammalian hormone.
Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase
Authors: Gahl W A , Vale A M , Pitot H C
Published: August 2015

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.
Purification and properties of the assimilatory nitrite reductase from barley Hordeum vulgare leaves
Authors: Serra J L , Ibarlucea J M , Arizmendi J M , Llama M J
Published: August 2015

The assimilatory nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) from barley (Hordeum vulgare L.) leaves has been purified over 1500-fold with a recovery of 30% and a specific activity of 84 mumol of nitrite reduced/min per mg of protein. The purification procedure includes (NH4)2SO4 fractionation, ion-exchange and molecular-sieve chromatographies and, finally, ferredoxin-Sepharose-4B affinity chromatography. The enzyme appears homogeneous by polyacrylamide gel electrophoresis and consists of a single polypeptide chain with an Mr of 61 000. The absorption spectrum of the pure enzyme was typical of a haem-containing protein. The enzyme showed low thermostability and was specific for ferredoxin (Km 0.4 microM), although reduced Methyl Viologen (Km 120 microM) was also effective. The same Km value for nitrite (250 microM) was obtained with both electron carriers. Cyanide acted as a powerful pure competitive inhibitor of enzyme with respect to nitrite (Ki 40 microM). Thiol-blocking agents also caused considerable inhibition, but only the ferredoxin-driven activity was significantly inhibited by sulphite and hydroxylamine.
The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding
Authors: Price N C , Stevens E
Published: August 2015

A number of aspects of the refolding of denatured rabbit muscle creatine kinase have been studied. Addition of substrates has no effect on the rate or extent of regain of activity. The changes in protein fluorescence during refolding broadly parallel the regain of activity. A study of the susceptibility of the enzyme to proteolysis during refolding indicates that there is no significant accumulation of folded, but inactive, intermediates in the folding process. Modification of the reactive thiol group on each subunit of the enzyme by small reagents such as iodoacetate or iodoacetamide prior to denaturation has only a small effect on the rate of subsequent refolding. However, modification by the bulky reagent 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate has a very large effect on the ability of the enzyme to refold after denaturation.
Brain clathrin and clathrin-associated proteins.
Authors: Lisanti M P , Schook W , Moskowitz N , Ores C , Puszkin S
Published: August 2015

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.
Cytoplasmic RNA sequences complementary to cloned chick δ-crystallin cDNA show size heterogeneity.
Authors: Bower D J , Errington L H , Wainwright N R , Sime C , Morris S , Clayton R M
Published: August 2015

Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes.
The slow kinetic transients of arylsulphatase A.
Authors: O'Fagain C , Bond U , Orsi B A , Mantle T J
Published: August 2015

A simple model is described to account for the anomalous time course of arylsulphatase A. In the case of the ox liver and human placental enzymes the enzyme-nitrocatechol sulphate complex can, in addition to forming products, slowly break down to form an inactive species which can turn in slowly regenerate active enzyme. When the inactive form binds sulphate the rate of reactivation is enhanced, 218-fold in the case of the ox enzyme. Rat liver arylsulphatase A is refractory to reactivation by sulphate.
The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.
Authors: Bert J L , Mathieson J M , Pearce R H
Published: August 2015

Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma.
Interaction of the pBR 322-coded RTEM α-lactamase with substrates. Evidence for specific conformational transitions.
Authors: Citri N , Zyk N
Published: August 2015

The rate of inactivation of RTEM-1 beta-lactamase by Pronase is accelerated by class A (‘resistant’) penicillins. Other substrates (class S penicillin and cephalosporins) protect against the inactivation. Cefoxitin, a semi-synthetic cephamycin, induces a more extensive, hysteretic response. In its presence the enzyme is inactivated by trypsin as well as by Pronase.
Kinetic studies on the reduction of cytochrome c. Reaction with dihydroxy conjugated compounds (catechols and quinols)
Authors: Saleem M M M , Wilson M T
Published: August 2015

The kinetics of reduction of cytochrome c by catechol(s), quinol(s) and related compounds were investigated by stopped-flow spectrophotometry. Studies on the influence of pH on the rates indicate that only deprotonated forms of these compounds act as reducing agents, with the dianionic forms being the most effective. The pH-independent second-order rate constants are reported. Hammett treatment of the effects of substituents on the aromatic ring structure of the reductants show that for electron transfer to occur the charge on the deprotonated species must not be withdrawn on to the substituents. Possible sites for electron donation to cytochrome c are discussed, and the results indicate that the haem edge is a likely candidate.
The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae
Authors: Philippe M. , Schrével J.
Published: August 2015

Gregarines, which are parasitic protozoa living in invertebrates, possess a cortical structure specific to their vegetative stage: namely two additional cytomembranes are lying just under the plasma membrane. This cortical complex has been isolated by centrifugation on discontinuous sucrose gradients and characterized chemically. Its integrity was tested by electron microscopy. Ghost proteins were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. About 30 polypeptides of mol.wt. 15000–300000 were present in this fraction and four glycoproteins were detected after periodate/Schiff staining. Ten major proteins were labelled after lactoperoxidase-catalysed iodination. The GP2 glycoprotein (41000–49000 apparent mol.wt.) appears to be a major component of the cell surface. Effects of trypsin and Pronase digestion on ghosts and cells were monitored by gel electrophoresis and by electron microscopy. Ghosts treated with low trypsin or Pronase concentrations (10–25μg/ml) became drastically disorganized; many proteins were vigorously attacked in comparison with those of control ghosts. Variations in proteinase-sensitivity of proteins are pointed out. The GP3 glycoprotein (130000–160000 apparent mol.wt.) seemed to be the only glycoprotein released from the cell surface by trypsin. Whole cells treated under the same conditions or with higher proteinase concentrations (up to 1mg/ml) do not exhibit morphological modifications of the cell surface; furthermore, no discernible cleavage of membrane proteins was indicated by electrophoretograms. It is postulated that cell-surface proteins are protected by the dense carbohydrate cell coat. By using various different methods (change of ionic strength, detergent, denaturing agent, labelling experiment) it was possible to localize several major proteins within the protozoon cortical membranes.
The effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain
Authors: Bause Ernst , Legler Günter
Published: August 2015

Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[14C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase in normal and dystrophic hamsters
Authors: Greenough M C , Boegman R J
Published: August 2015

The activity and diurnal variation of 3-hydroxy-3-methyglutaryl-CoA reductase (EC 1.1.1.34; HMG-CoA reductase), the rate-limiting enzyme in the cholesterol-biosynthetic pathway, of normal and dystrophic hamsters was determined. Liver enzyme activity showed a diurnal pattern in the normal male, but not in the dystrophic male. Enzyme values in normal males at the midpoint of the 12 h dark period were 10 times those in dystrophic males. No evidence for diurnal variation in the HMG-CoA reductase of the brain was observed, and similar activities were found for normal and dystrophic animals. The apparent Km for HMG-CoA reductase from the liver of normal or dystrophic hamsters was approx. 9 microM, and the Vmax. was 5.9 and 21.7 pmol/min per mg of protein for dystrophic and normal hamsters respectively.
The binding of platinum complexes to tuna cytochrome c
Authors: Boswell A P , Moore G R , Williams R J P
Published: August 2015

The binding of [PtCl4]2- and cis-[PtCl2(NH3)2] to methionine-65 of tuna cytochrome c was investigated by 1H n.m.r. The modification at methionine-65 is shown to cause an extremely small structural perturbation to the protein at the site of modification.
A continuous spectrophotometric determination of hepatic microsomal azo reductase activity and its dependence on cytochrome P-450
Authors: Mallett A K , King L J , Walker R
Published: August 2015

1. A continuous spectrophotometric determination of rat hepatic microsomal anaerobic azo reductase activity has been developed. 2. The addition of soluble flavins (riboflavin, FMN or FAD) greatly increased this NADPH-dependent activity towards a number of azo substrates. 3. Investigations with amaranth as substrate gave an apparent Km of 34 microM and Vmax. of 4 nmol/min per mg of microsomal protein. The inclusion of a fixed concentration of FMN increased Vmax. and greatly decreased Km, the magnitude of these changes reflecting the concentration of flavin present. 4. Investigations using a fixed amaranth concentration over a range of flavin concentrations gave biphasic double-reciprocal plots with two apparent Km and Vmax. values. 5. Pretreatment of animals with cobaltous chloride, 2-allyl-2-isopropylacetamide, carbon tetrachloride, phenobarbitone and 3-methylcholanthrene altered azo reductase activity in parallel with changes in cytochrome P-450 content. 6. The significance of these results is discussed in terms of the electron-transfer components present in the hepatic microsomal fraction.