bluuebunny/crossref_metadata_2025 huggingface dataset:
The functional significance of variants of fructose 1,6-diphosphatase in the gill and hypodermis of a marine crustacean. A kinetic study
Authors: Hochachka P. W.
Published: August 2015

1. In the hypodermis and gill of the Crustacea fructose 1,6-diphosphatase (EC 3.1.3.11) functions at a primary branch point between glycogen and chitin synthesis. In these tissues of the Arctic king-crab, Paralithodes camtchatica, fructose diphosphatase occurs in two electrophoretically distinguishable forms. 2. Fructose diphosphatase I (pI7.2–7.5) accounts for 70 and 10% of total fructose diphosphatase activity in the hypodermis and gill respectively, whereas fructose diphosphatase II (pI5.3) accounts for 30 and 90% of the total activity in the two tissues. Both forms display a neutral pH optimum, have an absolute requirement for a bivalent cation, and are potently inhibited by high concentrations of AMP and substrate. 3. Fructose 1,6-diphosphate saturation follows Michaelis–Menten kinetics for both fructose diphosphatases; the Km (fructose diphosphate) for fructose diphosphatase I is somewhat higher than for fructose diphosphatase II. In the presence of 50–200mm-K+, the Km (fructose diphosphate) increases and at high concentrations of K+ fructose diphosphate saturation follows sigmoidal kinetics. 4. UDP-N-acetylglucosamine and UDP-glucose at high concentrations specifically and potently inhibit fructose diphosphatase II, but do not significantly affect fructose diphosphatase I activity. 5. Low concentrations of UDP-N-acetylglucosamine activate fructose diphosphatase II by a decrease in the apparent Km (fructose diphosphate), but fructose diphosphatase I is again refractory to UDP-N-acetylglucosamine under these conditions. 6. In the presence of K+ and UDP-N-acetylglucosamine, fructose diphosphatase II is able to compete for limiting fructose diphosphate about three times more effectively than is fructose diphosphatase I. 7. AMP inhibition of both forms of the enzyme is subject to three independent variables: (a) alkaline pH increases the Ki (AMP), (b) K+ decreases the Ki, increases the sigmoidicity of inhibition kinetics, increases the maximum inhibition attained, and abolishes the effect of pH on AMP inhibition, and (c) Mg2+ strongly de-inhibits AMP-inhibited fructose diphosphatase. 8. It is postulated that the presence of two forms of fructose diphosphatase aids controlled channelling of carbon through the fructose diphosphatase `bottleneck' either towards glycogen synthesis or chitin synthesis, but not towards both simultaneously.
The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile
Authors: Gordon Ellen R. , Chan Tak-Hang , Samodai Karen , Goresky Carl A.
Published: August 2015

Bilirubin and its conjugates were extracted from either dog gall-bladder bile or bile-containing human duodenal juice into chloroform containing 10mm-tetraheptylammonium chloride. The intact bilirubin tetrapyrroles were then separated by t.l.c. Structural elucidation was made after coupling of the individual pigments with diazonium salts. Four azopigments were detected: azopigment αo or dipyrrolic azobilirubin; azopigment δ or dipyrrolic azobilirubin monoglucuronide; azopigment α3 or dipyrrolic azobilirubin monoglucoside; and, from dog gall-bladder bile, azopigment α2. The last conjugate required further verification of its structure. After methanolysis, it was shown by combined g.l.c.–mass spectrometry to contain xylose in a 1:1 molar ratio with the azopigments of bilirubin. Human bile contained 86% bilirubin diglucuronide, 7% bilirubin monoglucuronide monoglucoside diester, 4% bilirubin monoglucuronide and 3% bilirubin. Dog gall-bladder bile had a considerably different composition; it contained 47% bilirubin diglucuronide, 40% bilirubin monoglucuronide monoglucoside diester, 8% bilirubin monoglucuronide, 4% bilirubin diglucoside, 1–2% bilirubin and traces of conjugates containing xylose. The total bilirubin content and proportions of the conjugates did not change in bile that was frozen and stored at −20°C under N2, whereas in the chloroform/tetraheptylammonium chloride extract, similarly stored, total pigment was slowly lost and the diglucuronide conjugate converted into the monoglucuronide.
Griseofulvin-induced aggregation of microtubule protein
Authors: Roobol A , Gull K , Pogson C I
Published: August 2015

Griseofulvin (7-chloro-2′,4,6-trimethoxy-6′-methylspiro[benzofuran-2(3H),1′-[2]cyclohexene]-3,4′-dione) induces aggregation of microtubule protein at 0 degrees C. This aggregate contains approx. 90% of the microtubule-associated proteins originally present in the microtubule protein. The supernatant obtained after removal of the griseofulvin-induced aggregate does not form microtubules on warming at 37 degrees C. Addition of the griseofulvin-aggregated protein to this supernatant and warming to 37 degrees C gives rise to a limited amount of microtubule assembly. The possible involvement of griseofulvin-induced aggregation of microtubule protein at 0 degrees C in the inhibition by griseofulvin of microtubule assembly in vitro is discussed.
Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase
Authors: Stinson R A
Published: August 2015

The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible ‘tightening’ of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.
Tritium effect in peroxidation of ehtanol by liver catalase
Authors: Damgaard S E
Published: August 2015

1. Simultaneous determination of the rate of appearance of 3H in water from [(1R)-1-3H1] ethanol and the rate of acetaldehyde formation in the presence of rat or ox liver catalase under conditions of steady-state generation of H2O2 allowed calculation of the 3H isotope effect. The mean value of 2.52 obtained for rat liver catalase at 37 degrees C and pH 6.3-7.7 was independent of both ethanol concentration and the rate of H2O2 generation over a wide range. At 25 degrees C a slightly lower mean value of 2.40 was obtained with the ox liver catalase. 2. Neither the product, acetaldehyde, nor 4-methylpyrazole influenced the two rates measured in the assay. 3. Relating the value obtained for the 3H isotope effect to a known value for the 2H isotope effect strongly supports the view that both values are close to the true isotope effect with the respective substituted compounds on the rate constant in the catalytic step involving scission of the C-H bond. 4. The constancy of the isotope effect under various conditions makes it possible to use it for interpretations in vivo. 5. It was established that beta-D-galactose dehydrogenase exhibits B-specificity towards the nicotinamide ring in NAD.
Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers
Authors: Watson D H , Wootton J C
Published: August 2015

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2′-phosphate-Sepharose 4B, is described. The assembly of the 2′-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2′-AMP and its derivatives, 8-bromoadenosine 2′-phosphate and 8-(l-aminohexyl)aminoadenosine 2′-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2′-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.
Interaction of lipoprotein lipase with heparin–Sepharose. Evaluation of conditions for affinity binding
Authors: Bengtsson Gunilla , Olivecrona Thomas
Published: August 2015

Lipoprotein lipases from a variety of sources have been shown previously to bind to heparin and some related polysaccharides. For the present studies lipoprotein lipase purified from bovine milk was used. 1. In batch experiments binding of the enzyme activity to heparin–Sepharose occurred relatively slowly, so that 30min was required for the system to come to near-equilibrium. In contrast, release of the enzyme activity from heparin–Sepharose by addition of salt to the liquid phase occurred rapidly. 2. Some binding was observed also with unsubstituted Sepharose, but this binding had a low capacity compared with that observed with heparin–Sepharose. High salt concentrations, heparin or deoxycholate decreased the binding to unsubstituted Sepharose. These factors also increase the solubility of the enzyme, which is low. 3. Addition of heparin to the liquid phase caused a concentration-dependent release of enzyme activity from the gel. These results suggested that the binding of the enzyme to heparin–Sepharose was mainly through interaction with heparin. 4. The enzyme activity was also quantitatively displaced to the liquid phase at increased concentrations of salt. Among the positive ions tested the following order of effectiveness was noted: Cs+≃K+>Na+>Li+; and among the negative the following: SCN−>I−> NO3−>Br−≃Cl−. The differences were quite large. Thus addition of 0.16m-KSCN (in addition to the 0.32m-NaCl originally present) displaced one-half of the enzyme activity to the supernatant, whereas 0.8m-LiCl only displaced one-quarter. 5. The distribution of heparin in the gel also profoundly influenced the binding. Two series of gels were studied. One series was made by mixing heparin–Sepharose with unsubstituted Sepharose. Results obtained with these gels were those expected from a series of decreasing volumes of heparin–Sepharose. In contrast, a series of heparin–Sepharoses made with different degrees of substitution gave quite different results. With these gels the amount of enzyme activity bound per amount of heparin increased markedly, whereas the salt concentration needed to displace the enzyme activity from the gel decreased markedly with decreased concentration of heparin in the gel. 6. On stepwise elution of small columns of heparin–Sepharose the enzyme activity was eluted over a remarkably wide range of salt concentrations. When enzyme eluted at one salt concentration was re-applied, it gave the same elution profile as enzyme previously eluted at other salt concentrations or the entire enzyme preparation. These and other results suggested that, whereas the enzyme preparation was rather homogeneous in its binding to heparin, the heparin preparation was polydisperse in binding of lipoprotein lipase.
Purification and properties of myosin light-chain kinase from fast skeletal muscle
Authors: Pires E M V , Perry S V
Published: August 2015

1. A procedure is described for the isolation of myosin light-chain kinase from rabbit fast skeletal muscle as a homogeneous protein. 2. Myosin light-chain kinase is a monomeric enzyme of mol.wt. 77000. Under some conditions of storage it is converted into components of mol.wts. about 50000 and 30000 that possess enzymic activity. 3. The enzyme is clearly different in structure and properties from any other protein kinase so far isolated from muscle. 4. The enzyme is highly specific for the P-light chain (18000-20000-dalton light chain) of myosin and requires Ca2+ for activity. 5. The P-light chain is phosphorylated at a similar rate whether isolated or associated with the rest of the myosin molecule. 6. The effects of pH, bivalent cation and other nucleotides on the enzymic activity are described. 7. The role of the phosphorylation of the P-light chain of myosin in muscle function is discussed.
Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae
Authors: Raziuddin S
Published: August 2015

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.
Solvent accessibilities in glycyl, alanyl and seryl dipeptides
Authors: Manavalan P , Ponnuswamy P K , Srinivasan A R
Published: August 2015

Theoretical studies on glycyl-alanyl and seryl dipeptides were performed to determine the probable backbone and side-group conformations that are preferred for solvent interaction. By following the method of Lee & Richards [(1971) J. Mol. Biol. 55, 379-400], a solute molecule is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, and a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the surface accessible to the solvent molecule, and hence the solvent accessibility for a particular conformation of the solute molecule, is computed. From the calculated solvent accessibilities for various conformations, solvation maps for dipeptides were constructed. These solvation maps suggest that the backbone polar atoms could interact with solvent molecules selectively, depending on the backbone conformation. A conformation in the right-handed bridge (zetaR) region is favoured for both solvent interaction and intrachain hydrogen-bonding. Also the backbone side-chain hydrogen-bonding within the same dipeptide fragment in proteins is less favoured than hydrogen-bonding between side chain and water and between side chain and atoms of other residues. Solvent accessibilities suggest that very short distorted alphaR-helical and extended-structural parts may be stabilized via solvent interaction, and this could easily be possible at the surface of the protein molecules, in agreement with protein-crystal data.
Kinetic studies of the phenol sulphate-phenol sulphotransferase of Aspergillus oryzae
Authors: Burns G R J , Galanopoulou E , Wynn C H
Published: August 2015

Arylsulphatase II of Aspergillus oryzae exhibits both hydrolytic and sulphotransferase activities. The kinetic data suggest the formation of an intermediate covalent enzyme-sulphate complex with transfer of sulphate from donor to acceptor proceeding via a Ping Pong mechanism. The unusual kinetic behaviour when 2-hydroxy-5-nitrophenyl sulphate is the substrate is also consistent with this mechanism.
The subunit and polypeptide-chain structure of rabbit secretory immunoglobulin A. Isolation of a proteolytic fragment suitable for sequence studies on the variable region of α-chain
Authors: Johnstone A P , Mole L E
Published: August 2015

A method was developed for the preparation of a proteolytic fragment of rabbit secretory immunoglobulin A (sIgA) which contains the variable region of the alpha-chain; this fragment is suitable for primary-sequence studies. The serologically defined subclasses of sIgA are shown to correlate partially with the nature of the binding of a constituent chain of sIgA, called secretory piece. Data are also presented on the relative resistance of sIgA to enzymic and reductive cleavage, compared with immunoglobulin G.
Sequence studies on the heavy chain of rabbit immunoglobulin A of different a-locus allotypes
Authors: Johnstone A P , Mole L E
Published: August 2015

The amino acid sequence was determined of part of the variable region of heavy chain from rabbit immunoglobulin A of allotypes a1 and a3. Two corrections of the primary sequence of Aa1 gamma-chains are reported; most of the structural correlates of the alpha-locus allotypes are confirmed. The amino acid sequence of the N-terminal 20 residues of alpha-negative molecules was also determined and found to be homologous to the human VhIII subgroup. These molecules are present in a much higher proportion in the alpha-chain pool than in the gamma-chain.
Adenosine triphosphatase and adenosine diphosphate/adenosine triphosphate isotope-exchange activities of the chromaffin-granule membrane
Authors: Apps D K , Reid G A
Published: August 2015

The association of adenosine triphosphatase and ADP/ATP isotope-exchange activities with chromaffin-granule membranes was shown by sucrose-density-gradient centrifugation. The two activities were solubilized, and separated by differential sedimentation.
Variation in the distribution of two human heart ferritin species. Isoferritin profile and subunit composition in normal and iron-overloaded subjects
Authors: Bomford A , Lis Y , McFarlane I G , Williams R
Published: August 2015

On polyacrylamide slab electrophoresis two ferritin species, termed fast and slow, were present in three control and four iron-loaded human hearts. The ratio of the fast to slow species varied between hearts and correlated with the distribution of isoferritins, which had pI values ranging from 4.8 to 5.6. The acidic isoferritins were prominent in all hearts, but one control and the four iron-loaded hearts contained, in addition, basic isoferritins. All ferritins were composed of two subunits, an acidic H type and a more basic HL type, which was the main subunit in normal liver. Hearts with the highest proportions of acidic isoferritin contained the highest proportion of the H subunit.
The sites of phosphorylation of rabbit cardiac troponin I by adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Effect of interaction with troponin C
Authors: Moir A J G , Perry S V
Published: August 2015

1. Troponin I prepared from rabbit hearts contains 1.0-1.5 mol of P/mol when isolated by affinity chromatography. Most of the covalently bound phosphate is located in residues 1-48 of the molecule. 2. 3′:5′-Cyclic AMP-dependent protein kinase catalyses phosphorylation at serine-20 and serine-146. Serine-20 is more rapidly phosphorylated than serine-146. 3. In troponin I prepared from frozen hearts by affinity chromatography about 0.3-0.5 mol of P/mol is associated with serine-20 and 0.8-1.0 mol of P/mol with other site(s) in residues 1-48 of the molecule. 4. Phosphorylation at serine-20 and servine-146 is not significantly inhibited by troponin C. 5. The mechansim of the interaction of troponin C with cardiac troponin I is discussed in the light of these results.
The effect of bovine tendon glycoprotein on the formation of fibrils from collagen solutions
Authors: Anderson J C , Labedz R I , Kewley M A
Published: August 2015

The formation of collagen fibrils under physiological conditions of ionic strength, pH and temperature was markedly affected by the presence of small amounts of bovine tendon glycoprotein. The absorbance of the gels at 400 nm was decreased, and they took longer to form. Over the range of concentration tested, the negative specific absorbance, -delta Asp., and the specific retardation, Rsp., both increased with the glycoprotein to collagen ratio. When added during the nucleation phase, glycoprotein was still able to exert its effect almost fully, and so must act to inhibit the later stages of fibril formation. Several pieces of evidence showed that glycoprotein acts via a weak binding to the collagen molecule. Electron microscopy established that fibrils formed in the presence of glycoprotein had a normal cross-striation pattern, but were significantly thinner than fibrils formed in control gets. The results suggest that glycoprotein could act in tissues to help regulate the diameter of collagen fibrils.
A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum
Authors: Main A. Russell , McKnelly Susan C. , Burgess-Miller Sybil K.
Published: August 2015

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18μm, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low kcat. of the monomeric enzyme, which was approx. 57s−1 with butyrylthiocholine as substrate and is one-thirtieth of the comparable kcat. of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH4)2SO4 and ion-exchange chromatography.
Inactivation of α- and β-thrombin by antithrombin-III, α2-macroglobulin and α1-proteinase inhibitor
Authors: Machovich R , Borsodi A , Blaskó G , Orakzai S A
Published: August 2015

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.
Activation studies of the multiple forms of prochymosin (prorennin)
Authors: Asato N , Rand A G
Published: August 2015

Activation of the four separate components of prochymosin (prorennin) at pH 5.0 demonstrated that each zymogen was the precursor to an electrophoretically distinct chymosin (rennin). When the increase in milk-clotting activity with time was analysed, the mechanism of activation of unfractionated prochymosin, individual prochymosin components, and a mixture of the prochymosin fractions at pH 5.0 was shown to follow essentially autocatalytic kinetics. The activation of prochymosin C was completed in 70 h, whereas the other three fractions each required more than 110 h for complete activation under the same conditions. Intact prochymosin, the mixture of four components and prochymosin C were activated at similar rates. Interaction of the individual fractions during activation is suggested to explain the increased rate of the activation for the mixture. Comparison of autocatalytic activation of unfractionated prochymosin purified chromatographically at pH 6.7 and 5.7 demonstrated an increased rate of reaction of the zymogen prepared at the lower pH value. The possibility that prochymosin became susceptible to activation during preparation at pH values slightly below 6.0, as a result of changes in the proportion of the components or a conformational change and exposure of the active site, is discussed.
Electron-paramagnetic-resonance studies of leghaemoglobins from soya-bean and cowpea root nodules. Identification of nitrosyl-leghaemoglobin in crude leghaemoglobin preparations
Authors: Maskall C. Sidney , Gibson John F. , Dart Peter J.
Published: August 2015

1. Leghaemoglobins from soya-bean (Glycine max) and cowpea (Vigna unguiculata) root nodules were purified by chromatography on DEAE-cellulose phosphate columns at pH8.0 and pH5.8, to avoid the relatively low pH (5.2) commonly used to purify these proteins. 2. E.p.r. (electron-paramagnetic-resonance) spectra of the fluoride, azide, hydroxide and cyanide complexes of these ferric leghaemoglobins were very similar to the spectra of the corresponding myoglobin derivatives, indicating that the immediate environment of the iron in leghaemoglobin and myoglobin is similar, an imidazole moiety of histidine being the proximal ligand to the haem iron [cf. Appleby, Blumberg, Peisach, Wittenberg & Wittenberg (1976) J. Biol. Chem.251, 6090–6096]. 3. E.p.r. spectra of the acid-metleghaemoglobins showed prominent high-spin features very near g=6 and g=2 and, unlike myoglobin, small low-spin absorptions near g=2.26, 2.72 and 3.14. The width of the g=6 absorption derivative at 10–20K was about 4–4.5mT, similar to the value for acid-methaemoglobin. In contrast, a recently published (Appleby et al., 1976) spectrum of acid-metleghaemoglobin a had less high-spin character and a much broader absorption derivative around g=6. 4. E.p.r. spectra of ferric leghaemoglobin nicotinate and imidazole complexes suggest that the low-spin absorption near g=3.14 can be attributed to a trace of ferric leghaemoglobin nicotinate, and those near g=2.26 and 2.72 are from an endogenous dihistidyl haemichrome. 5. A large e.p.r. signal at g=2 in all samples of crude leghaemoglobin was shown to be from nitrosyl-leghaemoglobin. A soya-bean sample contained 27±3% of the latter. A previously unidentified form of soya-bean ferrous leghaemoglobin a was shown to be its nitrosyl derivative. If this is not an artifact, and occurs in the root nodule, the nitrosyl radical may interfere with the function of leghaemoglobin.
The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa
Authors: Parr S R , Barber D , Greenwood C , Brunori M
Published: August 2015

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.
A simple radioassay for angiotensin-converting enzyme
Authors: Ryan J W , Chung A , Ammons C , Carlton M L
Published: August 2015

Angiotensin-converting enzyme can be measured by the rate of release of 3H-labelled hippurate from p-[3H]benzoylglycylglycylglycine. The product is separable from the substrate by extraction of acidified reaction mixtures with ethyl acetate. Assay results for human serum angiotensin-converting enzyme can be obtained within 1.5 h of receipt of serum samples. Within the limits tested, the assay appears to be specific. However, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out.
Chemical modification of cellulase from Aspergillus niger
Authors: Hurst P L , Sullivan P A , Shepherd M G
Published: August 2015

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.
The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation
Authors: Roughley P J
Published: August 2015

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.
Purification, properties and cellular localization of the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena
Authors: Matcham G W J , Dodgson K S , Fitzgerald J W
Published: August 2015

The availability of homogeneous samples of the potassium salts of L- and D-octan-2-yl sulphate has enabled the separation of the optically stereospecific CS1 and CS2 secondary alkysulphohydrolases from extracts of cells of Comamonas terrigena. The CS2 enzyme was purified to homogeneity, and an initial study was made of its general properties, specificity, cellular localization and relationship to the CS1 enzyme. The CS2 enzyme has a molecular weight of approx. 250000 and a subunit size of approx. 58000, indicating that the molecule is a tetramer. Under the experimental conditions used the enzyme appears to be specific for (+)-secondary alkyl sulphate esters with the sulphate group at C-2 and with a chain length of at least six carbons. Enzyme activity towards racemic C-2 sulphates increases with increasing chain length up to C10, and there is some indirect evidence to suggest that activity declines when that chain length is exceeded. Other indirect evidence confirms that the CS1 enzyme exhibits similar specificity, except that only (-)-isomers can serve as substrates. Both enzymes are present in broth-grown stationary-phase cells of C. terrigena in approximately equal amounts.
The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule
Authors: Thorley-Lawson David A. , Green N. Michael
Published: August 2015

The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[14C]maleimide or 5,5′-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[14C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A1 and A2). The kinetics of the reaction of the ATPase with 5,5′-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca2+ the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca2+ the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca2+ of Mg2+ had very little effect on the reactivity.
The nature of the circular-dichoric spectra of complexes between ribonuclease A and nucleotides
Authors: Dudkin S M , Karabashyan L V , Karpeisky M Y , Mikhailov S N , Padyukova N S , Sakharovsky V G
Published: August 2015

The circular-dichroism and proton-magnetic-resonance spectra of complexes of ribonuclease A with dihydrouridine 3′-phosphate, 2′- and 3′-CMP, arabinosyl-3′-CMP, 1-(2-hydroxyethyl)cytosine 2′-phosphate and 1-(3-hydroxypropyl)cytosine 3′-phosphate were studied. Comparison of the results shows that non-additivity of the circular-dichroic spectrum of an enzyme-nucleotide complex may be due to: (a), alteration of the circular dichroic spectrum of the nucleotide under the influence of the asymmetric protein matrix (induced dichroism), and (b) a change in the nucleotide conformation. The contribution of each of the two factors was estimated to calculate the circular-dichoroic spectra of 2′-CMP and 3′-CMP in complex with ribonuclease A. 3′-CMP in this complex was characterized by negative circular dichroism in the long-wavelength absorption band of the nucleotide, whereas 2′-CMP was characterized by positive circular dichroism. Since both nucleotides in the complex are known to be in an anti conformation, it follows that even small changes in the conformation considerably modify the circular-dichroic spectrum of the nucleotide in complex with the enzyme.
The separation and characterization of marmoset kidney β-d-galactosidase and β-d-glucosidase
Authors: Pierce R J , Price R G
Published: August 2015

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.
Proteoglycan-degrading enzymes. A radiochemical assay method and the detection of a new enzyme cathepsin F
Authors: Dingle J T , Blow A M J , Barrett A J , Martin P E N
Published: August 2015

1. Polyacrylamide beads containing entrapped 35S-labelled proteoglycan molecules have been prepared. 2. The measurement of release of radioactivity provides an extremely sensitive assay for proteoglycan-degrading enzymes, including proteinases and hyaluronidase. 3. The amount of label released is a logarithmic function of enzyme concentration or time of incubation. Experiments were made in an attempt to explain this. 4. Assays were made by the new method at several pH values, and with the inclusion of inhibitors to identify the proteoglycan-degrading enzymes of rabbit ear cartilage. 5. A previously undescribed proteinase active against proteoglycan at pH4.5 but unaffected by pepstatin, was discovered. The enzyme was named cathepsin F, and was partially purified and characterized; it was detected in human articular cartilage.
Steady-state kinetic studies of the negative co-operativity and flip-flop mechanism for Escherichia coli alkaline phosphatase
Authors: Waight Roy D. , Leff Paul , Bardsley William G.
Published: August 2015

1. A study of variations in experimental error of velocity measurement with substrate concentration for alkaline phosphatase reveals that the standard error is not constant or strictly proportional to velocity, but obeys a more complex dependence. 2. By using an approach based on error estimates at each individual substrate concentration, we show that the double-reciprocal plots in general are curved, necessitating a high-degree rate equation. The curves are analysed according to a recent classification of possible curve shapes for the 3:3 function, which is shown to be the lowest-degree rate equation satisfying the experimental data. 4. Other workers have supposed the enzyme to follow Michaelis–Menten kinetics, and it is shown that this assumption is approximately true at low temperatures in the absence of phosphate. 5. A study of the effects of phosphate concentration, pH and temperature on the kinetics shows that there is a gradual alteration in curve shape with these experimental variables, resulting in an apparent reduction in degree under certain special conditions, and particularly at low temperature. 6. It is shown that the steady-state kinetics do not require a flip-flop or half-of-sites reactivity mechanism as claimed, and a mechanism is proposed, a rate equation calculated and an analysis attempted. 7. An analysis of the product-inhibition effects for a linked two-sited Uni Bi enzyme is given. Alterations of asymptotic double-reciprocal slopes and limiting (1/ν) intercepts with products is discussed, and it is shown how the theory of product inhibition can be extended to complex kinetic situations to extract information as to molecular mechanism. 8. Deviations from Michaelis–Menten kinetics are expressed in terms of the magnitude of the appropriate Sylvester resultants.
Phosphorus nuclear-magnetic-resonance studies of a transition-state analogue complex of creatine kinase
Authors: Milner-White E J , Rycroft D S
Published: August 2015

31P nuclear-magnetic-resonance spectra of MgADP bound to creatine kinase in the presence of creatine NO3- ions show that there are two non-identical forms of the bound nucleotide. The sites have different affinities for the nucleotide. MgADP at the high-affinity site is in slow exchange (k less than 125s-1 at 27 degrees C) with free MgADP.
Phospholipase A activity associated with membranes of human polymorphonuclear leucocytes
Authors: Franson R , Weiss J , Martin L , Spitznagel J K , Elsbach P
Published: August 2015

Homogenates of human polymorphonuclear leucocytes (granulocytes) contain a Ca2+-dependent phospholipase A with optimal activity pH7.0. This enzyme is membrane-bound and is enriched in crude cytoplasmic-granule fraction. Ratezonal centrifugation of the cytoplasmic-granule fraction demonstrates that the phospholipase A is associated not only with specific- and azurophilic-granule populations but also with an ‘empty’ vesicular fraction containing 85% of the total alkaline phosphatase activity of whole homogenate. Thus this phospholipase is associated with granule as well as with other cellular membranes of human granulocytes.
The mechanism of action of primary alkylsulphohydrolase and arylsulphohydrolase from a detergent-degrading micro-organism
Authors: Cloves J M , Dodgson K S , Games D E , Shaw D J , White G F
Published: August 2015

Previous studies have shown that secondary alkylsulphohydrolases from certain detergent-degrading micro-organisms are unusual esterases in that they catalyse fission of the C-O bond of the alkyl sulphate ester linkage. The position of bond fission catalysed by a primary alkylsulphatase and an arylsulphohydrolase present in Pseudomonas C12B has now been investigated. The primary alkylsulphatase behaved like the secondary alkylsulphohydrolases in cleaving the C-O bond of potassium heptan-1-yl sulphate. In contrast, the arylsulphohydrolase, in common with other similar enzymes previously studied, catalysed the fission of the O-S bond of potassium p-nitrophenyl sulphate.
The first step in the activation of chicken pepsinogen is similar to that of prochymosin
Authors: Keilova H , Kostka V , Kay J
Published: August 2015

Chicken pepsinogen was incubated at pH2.5 with pepstatin. The zymogen activated itself by a sequential mechanism and an intact peptide derived from residues 1-26 in the protein was released in the first step. This peptide was found to inhibit the milk-clotting activities of pig and chicken pepsins and calf chymosin but to different extents.
Purification and properties of human chorionic gonadotropin/lutropin receptor from plasma-membrane and soluble fractions of bovine corpora lutea
Authors: Khan F S , Rathnam P , Saxena B B
Published: August 2015

Plasma-membrane and soluble fractions containing human chorionic gonadotropin/lutropin receptor were prepared from bovine corpora lutea by ultracentrifugation. The plasma-membrane and soluble fractions were studied for physicochemical properties, salts and gangliosides. The receptor preparations obtained from the plasma-membrane purified individually by sucrose-density-gradient centrifugation, which resulted in a partial dissociation of the hormone-binding subunit from the intact functional receptor unit, which consists of both hormone-binding (regulatory) and adenylate cyclase-associated (catalytic) subunits. The fractions containing the functional receptor unit were further purified by gel filtration on Sepharose-6B and chromatography on concanavalin A-Sepharose. The ‘receptor’ was finally purified by affinity chromatography on a column of controlled-pore glass covalently coupled to hu man chorionic gonadotropin. The purified receptor from the plasma-membrane and the soluble fractions contained binding capacities of 901000 and 87000 fmol of human chorionic gonadotropin/mg of protein. Yields of 0.02 and 0.22mg of protein were obtained from 250 g of bovine corpora lutea, which represents a 10000- and 1000-fold increase respectively in the specific binding with 125I-labelled human chorionic gonadotropin. Immunization of rabbits with a partially purified receptor fraction generated antibodies that specifically inhibited the binding of the 125I-labelled human chorionic gonadotropin to the receptor.