bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improved purification of pyruvate decarboxylase from wheat germ
Authors: ZEHENDER Hartmut , TRESCHER Dorothea , ULLRICH Johannes 
Published: March 2005

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity‐staining, produced two bands of different molecular masses and chain compositions. The high‐molecular‐mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of Mr= 33000, as judged from sodium dodecyl sulfate electrophoreses. The low‐molecular‐mass band contained two types of chains with Mrα= 63000–65000 and Mrβ= 61000–62000. The N termini of both chains were threonine, whereas their C‐terminal sequences were different: α, ‐(Val)‐(Ser)‐(Ala)‐Leu; β, ‐(His)‐(Asp)‐(Ala)‐Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different.In all properties investigated, the low‐molecular‐mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2‐p‐toluidinonaphthalene‐6‐sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2‐Hydroxy‐5‐nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor‐binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.
Sequential processing reactions in the formation of hormone amides
Authors: BLEAKMAN Alexandra , SMYTH Derek G. 
Published: March 2005

The substrate specificity of an enzyme with amidating activity, present in porcine pituitary, was investigated by examining its ability to convert the synthetic peptides D‐Tyr‐Val‐Gly and D‐Tyr‐Val‐Gly‐Lys‐Arg to the dipeptide amide D‐Tyr‐Val‐CONH2. The purified enzyme catalysed the amidation reaction with the tripeptide but did not accept the pentapeptide as a substrate. With the mixture of enzymes present in a membrane fraction from porcine pituitary or the enzymes in a secretory granule fraction, both the tripeptide and pentapeptide substrates gave rise to D‐Tyr‐Val amide; the formation of dipeptide amide from the pentapeptide, however, involved a latency period after which amidation occurred at a similar rate with the two substrates. Evidence was obtained that arginine and lysine were released from the C terminus of the pentapeptide before amidation took place since the rate of formation of dipeptide amide was reduced at pH values that were compatible with amidation but unfavourable to the action of carboxypeptidase H. In addition formation of the dipeptide amide from the pentapeptide was blocked by guanidinoethylmercaptosuccinic acid and glycylarginine, which are inhibitors of carboxypeptidase enzymes.The experiments demonstrate that removal of basic residues from the C terminus of a peptide and amidation at C‐terminal glycine are reactions that take place consecutively. These prohormone‐processing reactions, which are intrinsic to the formation of hormone amides, did not synergise.
Computer‐graphics interpretations of residue exchanges between the α, β and γ subunits of human‐liver alcohol dehydrogenase class I isozymes
Authors: EKLUND Hans , HORJALES Eduardo , VALLEE Bert L. , JÖRNVALL Hans 
Published: March 2005

Three‐dimensional models of human alcohol dehydrogenase subunits have been constructed, based on the homologous horse enzyme, with computer graphics. All types of class I subunits (α, β, and γ) and the major allelic variants (β1/β2 and γ1/γ2) have been studied. Residue differences between the E‐type subunit of the horse enzyme and any of the subunits of the human isozymes occur at 64 positions, about half of which are isozyme‐specific. About two thirds of the substitutions are at the surface and all differences can be accommodated in highly conserved three‐dimensional structures.The model of the γ isozyme is most similar to the crystallographically analyzed horse liver E‐type alcohol dehydrogenase, and has all the funtional residues identical to those of the E subunit except for one which is slightly smaller: Val‐141 in the substrate pocket. The residues involved in coenzyme binding are generally conserved between the horse enzyme and the α, β, and γ types of the human enzyme. In contrast, single exchanges of these residues are the ones involved in the major allelic differences (β1 versus β2 and γ1 versus γ2), which affects the overall rate of alcohol oxidation since NADH dissociation is the rate‐determining step. Residue 47 is His in β2 and Arg in the β1, γ1, and γ2 subunits, and in horse liver alcohol dehydrogenase. Both His and Arg can make a hydrogen bond to a phosphate oxygen atom of NAD; hence the lower turnover rate of β1 apparently derives from a charge effect. The substitution to Gly in the α subunit results in one less hydrogen bond in NAD binding, and consequently in rapid dissociation. This may explain why the overall rate is an order of magnitude faster than that of β1. The important difference between γ1 and γ2 is an exchange at position 271 from Arg to Gln which can give a hydrogen bond from Gln in γ2 to the adenine of NAD. The tighter binding to γ2 can account for the slower overall catalytic rate in this isozyme.The kinetics and interactions of cyclohexanol and benzyl alcohol with the isozymes were judged by docking experiments using an interactive fitting program. Most of the differences in Km values for alcohols of the β and γ subunits could be related to one amino acid difference, i.e. Thr‐48 in the β subunit, compared with Ser in the γ and the horse liver enzyme subunits. The substrate pocket of our model of the α subunit differs most markedly both from those of the other subunits of the human isozymes and from that of the horse enzyme. Substitution of Ala‐93 in α for the Phe in the others makes the substrate cleft much wider close to the active‐site zinc atom and its ligand Cys‐174. Our model of the α subunit suggests that the substrate specificity of isozymes with this subunit differs significantly from that of other isozyme subunits.
Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species
Authors: MEIJER Johan , LUNDQVIST Gerd , De PIERRE Joseph W. 
Published: March 2005

Sex and species differences in hepatic epoxide hydrolase activities towards cis‐ and trans‐stilbene oxide were examined in common laboratory animals, as well as in monkey and man.In general trans‐stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude ‘mitochondrial’ fraction towards trans‐stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases.Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the ‘mitochondrial’ fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and ‘mitochondrial’ epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical.The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.
Are calcium‐dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes?
Authors: MIESKES Gottfried , KUDUZ Josko , SÖLING Hans‐Dieter 
Published: March 2005

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6‐phosphofructo‐1‐kinase from rat liver and rabbit muscle; pyruvate kinase, 6‐phosphofructo‐2‐kinase and fructose‐1,6‐bisphosphatase from rat liver) by defined concentrations of two cAMP‐independent protein kinases: Ca2+/calmodulin‐dependent protein kinase and Ca2+/phospholipid‐dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP‐dependent protein kinase. The following results were obtained.

Ca2+/calmodulin‐dependent protein kinase phosphorylates 6‐phosphofructo‐1‐kinase and L‐type pyruvate kinase at a slightly lower rate as compared to cAMP‐dependent protein kinase.
6‐Phosphofructo‐1‐kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L‐type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer.
Rabbit muscle 6‐phosphofructo‐1‐kinase is phosphorylated by cAMP‐dependent protein kinase but not by Ca2+/calmodulin‐dependent protein kinase.
Fructose‐1,6‐bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin‐dependent protein kinase when compared to the phosphorylation by cAMP‐dependent protein kinase.
6‐Phosphofructo‐2‐kinase is phosphorylated by Ca2+/calmodulin‐dependent protein kinase only at a negligible rate.
Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6‐phosphofructo‐2‐kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP‐dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different.

The results indicate that Ca2+/calmodulin‐dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP‐dependent protein kinase seem unlikely.
Characterization of epitopes on a variant surface glycoprotein from Trypanosoma congolense by six monoclonal antibodies
Authors: REINWALD Erwin , GREISER‐WILKE Irene , ARTAMA Wayan , RISSE Hans‐Jörg , MÖLLING Karin 
Published: March 2005

Monoclonal antibodies were isolated from mice immunized with variant surface glycoprotein of Trypanosoma congolense. Five out of the six monoclonals were able to detect epitopes at the cell surface in an indirect immunofluorescence analysis. One antibody did not react. Using protein‐A‐containing bacterial adsorbent all monoclonal antibodies precipitate glycosylated as well as non‐glycosylated variant surface glycoprotein. Carbohydrate chains therefore do not appear to be part of the immunodeterminant structure recognized by the various monoclonal antibodies.Interaction of the monoclonal antibodies with protein fragments obtained by partial proteolysis with V8 protease from Staphylococcus aureus or papain allows the classification of the antibodies into three groups with different epitope specificity.
Two hydrophobic low‐molecular‐mass protein fractions of pulmonary surfactant
Authors: CURSTEDT Tore , JÖRNVALL Hans , ROBERTSON Bengt , BERGMAN Tomas , BERGGREN Per 
Published: March 2005

Hydrophobic low‐molecular‐mass proteins were isolated from minced pig lungs and separated into two fractions. Electrophoresis of protein fraction 1 showed two major bands. Calculations of molecular masses from the electrophoretic mobilities are unreliable because of the extreme hydrophobicity of the peptides. However, the two bands were at positions corresponding to apparent molecular masses of about 3 kDa and 14 kDa, while sequence degradation disclosed only one major structure. Electrophoretic separation of protein fraction 2 revealed one band, at an apparent molecular mass of about 6 kDa. Microheterogeneities at the N terminus of both fractions were observed. However, the two fractions had different N‐terminal structures and amino acid compositions. Consequently they are concluded to represent different polypeptides without common segments. Bronchoalveolar lavage from humans also contains surfactant polypeptides and at least the fraction 2 peptide is highly similar in human and porcine surfactants.Artificial surfactant preparations, obtained by recombination of protein fraction 1 or 2 with a mixture of synthetic phospholipids, were evaluated with the pulsating bubble method and in experiments on artificially ventilated premature newborn rabbits. The addition of protein fraction 1 to the phospholipid mixture improved surface adsorption from more than 300 s to about 2 s and reduced minimum surface tension from more than 20 mN/m to nearly 0 as measured with a pulsating bubble. When this surfactant preparation was instilled into the airways of newborn rabbits, the tidal volumes at insufflation pressure 25 cm H2O was increased about twentyfold compared to the volumes obtained in non‐treated controls. Preparations based on protein fraction 1 had better in vitro and in vivo properties than those based on protein fraction 2. Both these protein‐based preparations were decidedly more effective than phospholipids alone.
Modification of flow‐force relationships by external ATP in yeast mitochondria
Authors: RIGOULET Michel , GUERIN Bernard , DENIS Michel 
Published: March 2005

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m‐chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP.ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m‐chlorophenylhydrazone in the presence of ATP.At the same respiratory rate, cytochrome a+a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome‐c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP.Control of respiration exerted by cytochrome‐c oxidase was measured under the three conditins mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions.These results suggest a regulatory effect of external ATP on cytochrome‐c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome‐c oxidase, on the other, depend respectively on the kinetic regulations of the system.
Characterization of a mannosidase acting on α1 → 3‐ and α1 → 6‐linked mannose residues of oligomannosidic intermediates of glycoprotein processing
Authors: MONIS Elizabeth , BONAY Pedro , HUGHES R. Colin 
Published: March 2005

Baby hamster kidney (BHK) cell extracts catalyze the conversion of [3H]mannose‐labelled (Man)5GlcNAc and (Man)6GlcNAc oligosaccharides to a (Man)3GlcNAc species that retains affinity for concanavalin‐A‐Sepharose and appears to be Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The properties of the (Man)5GlcNAc‐hydrolase activity differ from lysososomal α‐mannosidases as well as previously described processing mannosidases acting on oligosaccharide intermediates of N‐glycan assembly. Mosquito cell extracts catalyze hydrolysis of (Man)6GlcNAc but lack the (Man)5GlcNAc hydrolase activity detected in BHK cell extracts.Glycopeptide analysis has been carried out on a ricin‐resistant BHK mutant RicR14 that lacks N‐acetylglucosaminyl transferase I and fails to convert oligoigomannosidic N‐glycans to complex‐type chains, and mosquito cells that constitutively lack N‐acetylglucosaminyl transferase I. In both cells lines, the cellular glycoproteins contain (Man)5GlcNAc oligosaccharide as the major stable component equivalent to a 15–20‐fold increase compared with normal BHK cells. Although containing very high amounts of asparagine‐linked (Man)5(GlcNAc)2, RicR14 cells exhibit (Man)5GlcNAc hydrolase activity at levels similar to wild‐type BHK cells. This result, together with previous work [Foddy, L., Feeney, J. & Hughes, R. C. (1986) Biochem. J. 233, 697–706] showing the complete inhibition of conversion of oligomannosidic intermediates to complex‐type N‐glycans in BHK cells treated with swainsonine, an inhibitor of mannosidase II but not the (Man)5GlcNAc hydrolase activity, argues against a major role for the (Man)5GlcNAc hydrolase activity in N‐glycan asembly and suggesting other functions for the mannosidase activity in BHK cells.
Type 1, blue copper proteins constitute a respiratory nitrite‐reducing system in Pseudomonas aureofaciens
Authors: ZUMFT Walter G. , GOTZMANN Dany J. , KRONECK Peter M. H. 
Published: March 2005

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 ± 3 kDa). Its pl was 6.05.The EPR spectrum showed an axial signal with g‖ at 2.21(8) and g‖ at 2.04(5) The magnitude of the hyperfine splitting (A‖= 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (ɛ= 7.0 mM−1 cm−1), and a broad shoulder around 780 nm.A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (ɛ= 2.5 mM−1 cm−1) and pronounced structures in the ultraviolet region. The EPR parameters were g‖= 2.26(6) and g‖= 2.05(6), with A‖= 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide.The specific nitrite‐reducing activity with ascorbate‐reduced phenazine methosulfate as electron donor was 1 μmol substrate min−1 mg protein−1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite‐reduced methyl viologen and nitrite. No ‘oxidase’ activity could be demonstrated for the enzyme.Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomones.
The degree of branching in (α1,4)‐(α1,6)‐linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes
Authors: TOLMASKY Diana Silvia , KRISMAN Clara Rebeca 
Published: March 2005

Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan‐degrading enzymes.
In ‘sweet corn’ maize, two separate fractions with (α1,4)glucan: (α1,4)glucan α6‐glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above‐mentioned conditions.
Comparative analyses performed with native (α1,4)‐(α1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans.
It may be concluded that the branching enzyme is responsible for the specific degree of (α1,6) branch linkages found in the native polysaccharide.
The regulatory and basal phosphorylation sites of hormone‐sensitive lipase are dephosphorylated by protein phosphatase‐1, 2A and 2C but not by protein phosphatase‐2B
Authors: OLSSON Håkan , BELFRAGE Per 
Published: March 2005

The activity of hormone‐sensitive lipase, the rate‐limiting enzyme in adipose tissue lipolysis, is controlled by cAMP‐mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Strålfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317–3321]. The capacity of protein phosphatase‐1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase‐4 and cAMP‐dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (α subunit). Protein phosphatase‐1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone‐sensitive lipase, while protein phosphatase‐2B had no measureable activity towards any of the sites. When the activities of protein phosphatase‐1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4.Protein phosphatase‐1 showed 20% higher and protein phosphatase‐2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase‐2A and 2C, but relatively poor substrates for protein phosphatase‐1. Protein phosphatase‐2C activity towards the lipase was completely dependent on Mg2+ with a half‐maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase‐1 3–5‐fold with half‐maximal effect at 0.6 μg/ml, and by protein phosphatase‐2A about 2‐fold with half‐maximal effect at 3–5 μg/ml, thus illustrating the potential for control of these lipase phosphatase activities.