bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phase Transitions in the Membrane of a Marine Bacterium, Pseudomonas BAL‐31
Authors: TSUKAGOSHI Norihiro , PETERSEN Marianne H. , HUBER Ursula , FRANKLIN Richard M. , SEELIG Joachim 
Published: March 2005

An unsaturated fatty acid auxotroph, strain UFA, isolated from the marine pseudomonad Pseudomonas BAL‐31, host cell of the lipid‐containing bacteriophage PM2, was grown in media supplemented with different unsaturated fatty acids. Under these conditions the fatty acid composition of the cell could be altered drastically. The phase transition in the native membrane and in the extracted lipids was analyzed by electron spin resonance using a nitroxide spin probe. Membranes prepared from strain UFA grown in cisl6: 1 or trans16:1 showed one transition at 9.4 °C and 12.4 °C respectively. Extracted lipids in both cases had almost the same transition temperature as that of the intact membrane. Membranes prepared from Pseudomonas BAL‐31 had one transition at ∼ 12 °C, on the other hand there was no clear cut phase transition using extracted lipids.Replication of bacteriophage PM 2 took place below the transition temperature of the membrane lipids in the case where strain UFA was grown in trans 16: 1. Other cases were not studied.
On The Mechanism of Amylose Branching by Potato Q‐Enzyme
Authors: BOROVSKY Dov , SMITH Eric E. , WHELAN William J. 
Published: March 2005

When potato Q‐enzyme converts amylose into an amylopectin‐like molecule, the action is by a random, endo‐type transglycosylation of the substrate chains.
Inter‐chain transfer takes place during the formation of the amylopectin branch linkage. This is seen in experiments in which radioactive label was transferred between substrates of disparate molecular weight. Intra‐chain transfer, leading to the formation of a branch linkage, is not excluded by these experiments.
The minimum length of amylose chain that can act as an acceptor in the transglycosylation reaction, under the experimental conditions described, is greater than 40 glucose units.
The requirement of Q‐enzyme for substrate chains at least 40 glucose units in length is interpreted as meaning that a stabilized secondary and tertiary structure must be established in the substrate before it can be utilized by Q‐enzyme, and that the forces that provide such conformation are sufficiently strong only when the chains are longer than the minimum. Inter‐chain transfer is seen as taking place by one of two mechanisms. The first involves the reaction of the enzyme with a chain that has a stabilized (helical?) conformation. An enzyme‐donor chain intermediate is formed, that then reacts with an acceptor chain to complete the transglycosylation. The second mechanism envisages the substrate for the enzyme as being a complex formed between two chains (a double helix?). The enzyme encounters the complex and carries out an inter‐chain transglycosylation reaction.
Molecular‐Weight Determination of Animal‐Cell RNA by Electrophoresis in Formamide under Fully Denaturing Conditions on Exponential Polyacrylamide Gels
Authors: SPOHR Georges , MIRAULT Marc‐Edouard , IMAIZUMI Tereza , SCHERRER Klaus 
Published: March 2005

A method for electrophoretic analysis of RNA under fully denaturing conditions on exponential gradient polyacrylamide gels is described. Full denaturation, and strand separation of DNA · RNA hybrids and double‐stranded RNA is obtained in dry formamide only if electrophoresis is carried out at 45° and 55 °C, respectively.In such conditions, the effects of secondary structure of RNA, important in aqueous medium, are suppressed and a linear correlation is obtained between the logarithm of the molecular weight of an RNA and its final position in the gel over the entire molecular weight range of 104–107.Based on absolute molecular weight standards, obtained from sequenced rRNA of Escherichia coli and tRNA and extrapolating to higher molecular weights, the size of animal cell RNA was reexamined. Precursor rRNA (45 S) from HeLa cells migrates according to a molecular weight of 4.1 × 106. Nascent precursor mRNA has molecular weights of up to 5 × 106 in the case of duck erythroblasts and of up to 107 in HeLa cells. This seems to represent the largest size of non‐viral animal‐cell RNA molecules.
Studies on New Intracellular Proteases in Various Organs of Rats
Authors: KOMINAMI Eiki , KATUNUMA Nobuhiko 
Published: March 2005

Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37°C. Addition to the homogenate of coenzymes and the various keto‐acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of the enzyme.Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen‐antibody precipitates from homogenates containing inactivated enzyme. were separated by electrophoresis on sodium dodecylsulfatepolyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes.However, in vivo there was no difference between the rates of decrease of enzyme activity and of immunological activity during the decrease of ornithine aminotransferase following inhibition of protein synthesis. Moreover, no degradation products of the enzyme were found under either control, or pyridoxine‐deficient conditions. But the rate of decay of ornithine aminotransferase in the muscle layer of the small intestine was faster in pyridoxine deficiency. The apparent half‐life in vitamin‐B6‐depleted rats was estimated as 5 h, which was shorter than that in control rats (8 h). These results suggest that a step in the conversion of the holoenzyme to the apoenzyte is ratelimiting in degradation of the enzyme in vivo.
The Binding of Lipid to the Lipid‐Free Adenosine Triphosphatase Protein of Sarcoplasmic Reticulum
Authors: HARDWICKE Peter M. D. 
Published: March 2005

Dinitrophenylated dipalmitoyl phosphatidylethanolamine and its lyso derivative have been shown to bind to the lipid‐free ATPase protein derived from the sarcoplasmic reticulum. The binding of these lipids is accompanied by the quenching of up to 95% of the tryptophyl fluorescence of the protein. This effect is reversed by 9–10 mM deoxycholate.The solubility of the lipid‐free ATPase protein in the absence of deoxycholate and the solubility of submillimolar concentrations of the dinitrophenylated monopalmitoyl phosphatidylethanolamine anion in aqueous media allowed binding experiments using this lipid ligand to be carried out in a simple buffer system. It is shown that in the case of this lipid the initial phase of the binding process displays an apparent positive co‐operativity. Data from the second phase in the saturation of the protein with this lipid is consistent with binding to independent, equivalent, non‐interacting sites with a microscopic (intrinsic) association constant of 1.63 ×106 M−1, the fluorescence being quenched in a geometric fashion. Altogether a total of about 15 molecules of this lipid may be bound by the protein.
The Trypsin and Chymotrypsin Inhibitors in Chick Peas (Cicer arietinum L.)
Authors: BELEW Makonnen , EAKER David 
Published: March 2005

The two principal isoinhibitors P‐5 and P‐6 isolated earlier from the seeds of chick peas (Cicer arietinum L.) by a procedure involving biospecific affinity chromatography on active, matrix‐bound trypsin are shown to be the virgin and trypsin‐modified forms of the same inhibitor. The virgin inhibitor P‐5 consists of a single peptide chain of 66 amino acid residues cross‐linked by seven disulfide bridges. Upon interaction with the matrix‐bound trypsin under the conditions used the virgin inhibitor P‐5 is about 50% converted to P‐6 by cleavage of a single Lys‐Ser linkage at position 14– 15 in the molecule. The resulting tetradecapeptide remains bound to the rest of the molecule by two or four disulfide bridges, but the two fragments representing residues 1–14 and 15–66 separate readily by molecular‐sieve chromatography following reductive or oxidative cleavage of the disulfide linkages. The sequence of residues 1‐25 was established by Edman degradation.Cleavage of the Lys14–Ser15 linkage at the trypsin‐reactive site of the virgin inhibitor significantly reduces its trypsin inhibitory activity without affecting its anti‐chymotryptic activity. Upon treatment with carboxypeptidase B the trypsin‐modified inhibitor loses its anti‐tryptic activity but remains fully active against chymotrypsin. A similar treatment has no effect on the virgin inhibitor. Furthermore, the complex of the inhibitor with trypsin is inactive towards trypsin but remains fully active against chymotrypsin and vice versa. It is thus concluded that the principal inhibitor in chick peas inhibits both enzymes simultaneously and is clearly of the ‘double‐headed’ type with separate and independent reactive sites for the two enzymes. The molecule appears to be highly homologous to the Bowman‐Birk soybean inhibitor and to the lima bean inhibitor.
Antigenic Determinants of Murein Lipoprotein and Its Exposure at the Surface of Enterobacteriaceae
Authors: BRAUN Volkmar , BOSCH Valerie , KLUMPP Ernst Rudolf , NEFF Ingrid , MAYER Hubert , SCHLECHT Siegfried 
Published: March 2005

Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Sulmonellu, Arizona and Shigellu antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti‐lipoprotein titers than anti‐R‐lipopolysaccharide titers. Correspondingly, absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipopolysaccharide on the cell surface. In wild‐type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface.Antisera were prepared in rabbits against erythrocytes coated with various lipoprotein samples which differ at the C‐terminal end of the polypeptide chain. Antibodies against lipoprotein containing two attached muropeptides (called lipoprotein I) are directed against the muropeptide attachment region. Antibodies against lipoprotein ending with lysine‐55 (lipoprotein II) were directed specifically against this terminal lysine residue. No serological reaction was observable after splitting off this lysine residue by carboxypeptidase B. Remarkably this antiserum against lipoprotein II neither reacted with the free lipoprotein ending with lysine‐58 (lipoprotein IV) nor with the muropeptide containing lipoprotein I. Antisera against the lipoprotein lacking lysine‐55 (lipoprotein III) reacted with all lipoprotein samples as did the antibodies in antisera prepared against whole heat‐killed cells, thus indicating a common binding site located in the polypeptide chain towards the N‐terminal end. No indication of an antigenic reactivity of the co‐valently linked lipoid was obtained.Lipoprotein treated with boiling 4% sodium dodecylsulfate and acetone reacted strongly with antisera against the native lipoprotein. The recovery of the antigenic sites demonstrates the remarkable stability of the native conformation.The lipoprotein is a new common antigen for closely related Enterobacteriaceae. Lipoprotein antisera might be useful for characterization of outer membrane mutants of Enterobacreriaceue.
Conformational Changes Associated with Transient Activation of Phosphorylase in Glycogen Particles
Authors: BUSBY Stephen J. W. , GADIAN David G. , GRIFFITHS John R. , RADDA George K. , RICHARDS Rex E. 
Published: March 2005

Calcium‐dependent transient phosphorylation of phosphorylase b has been monitored in a rabbit muscle glycogen particle fraction. Using a phosphorus nuclear magnetic resonance assay, the changes in concentrations of small phosphate‐containing metabolites associated with this event have been measured. In addition, the conformation of phosphorylase has been monitored during transient activation by observing changes in the electron spin resonance signal from added spin‐labelled phosphorylase.
The transient activation was associated with a loss of glucose 6‐phosphate from phosphorylase b; newly formed phosphorylase a binds the nucleotides ADP, AMP or IMP. Because of the fast interconversion of these nucleotides the species bound to phosphorylase a change throughout the process.
Lowering the [Mg2+]: [Ca2+] ratio during transient activation causes accumulation of ADP. Electron spin resonance data from spin‐labelled phosphorylase shows that, under these conditions, ADP binding to phosphorylase a is potentiated.
Calcium‐dependent activation in the glycogen particle fraction is compared to the activation of phosphorylase in vivo.
Influence of Chain Length and Unsaturation on the Effects of Fatty Acids on Phosphoglyceride Biosynthesis in Isolated Rat and Pig Hepatocytes
Authors: ÅKESSON Björn , SUNDLER Roger , NILSSON Åke 
Published: March 2005

Hepatocytes isolated from rat or pig by collagenase perfusion were incubated with [3H]glycerol and different albumin‐bound fatty acids. Among C22 fatty acids docosahexaenoic acid stimulated phosphatidylethanolamine synthesis in rat hepatocytes most effectively. Addition of docosahexaenoic acid plus either palmitic or stearic acid resulted almost in the same stimulation whereas combinations of this acid with lauric or myristic acid had no effect. Lauric acid and myristic acid alone inhibited phosphatidylethanolamine synthesis. The chain length specificity for monoenoic fatty acids was similar, the hexadecenoic and octadecenoic acids (both cis and trans) being most stimulatory. The addition of 0.2 mM ethanolamine markedly stimulated phosphatidylethanolamine synthesis, but most effects of fatty acids were similar in its presence or absence.The effect of fatty acids on phosphatidylcholine and total lipid synthesis were often similar but clearly deviated from those on phosphatidylethanolamine synthesis.In isolated pig hepatocytes the effects of fatty acids were similar to those in the rat. The distribution of [3H]glycerol among molecular species of glycerolipids changed drastically upon fatty acid addition.The different effects on phosphatidylethanolamine and phosphatidylcholine synthesis are most probably due to differences in the substrate specificities of ethanolaminephosphotransferase and cholinephosphotransferase.
Lysis of Yeast Cell Walls Lytic β‐(1→6)‐Glucanase from Bacillus circulans WL‐12
Authors: ROMBOUTS Frank M. , PHAFF Herman J. 
Published: March 2005

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL‐12 produces wall‐lytic enzymes in addition to non‐lytic β‐(1→3)‐and β‐(1→6)‐glucanases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic β‐(1→6)‐glucanase and two lytic β‐(1→3)‐glucanases. The lytic β‐(1→6)‐glucanase was further purified by chromatography over diethylamino‐ethyl‐agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went through a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls was attributed to a debranching of the alkali‐insoluble β‐(1→3)‐glucan in the wall. Low molecular weight products from yeast cell walls included gentiotriose, gentiobiose and glucose but β‐(1→3)‐linked oligosaccharides were not detected. The lytic β‐(1→6)‐glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis‐Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro‐equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement.The lytic β‐(1→6)‐glucanase differs in essence from the non‐lytic β‐(1→3)‐glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.
Nuclease S1 Cleavage and the Primary Structure of Mitochondrial DNA
Authors: KOCH Jürgen , BRUHN Andreas 
Published: March 2005

The single‐strand‐specific nuclease S1 from Aspergillus oryzae rapidly converts superhelical mitochondrial DNA (African Green Monkey cells, Vero ATCC; CCL 81) into nicked circular DNA. These nicked mitochondrial DNA molecules contain two nicks, one in each strand. The phosphodiester backbones are cleaved during this reaction at or near sites that are alkali‐labile.In a second slow reaction the circular mitochondrial DNA is converted into a linear duplex DNA. Permutation tests indicate that this linear DNA represents a nonpermutated collection of DNA molecules.These results suggest that two of the alkali‐labile sites in the phosphodiester backbones of the mitochondrial chromosome are closely spaced on opposite strands and at specific positions.
Algal Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: O'BRIEN Michael J. , POWLS Roy 
Published: March 2005

Two enzymes with glyceraldehyde‐3‐phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion‐exchange chromatography and gel filtration.The D‐enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T‐enzyme has a molecular weight of 140000 and a VNADH: VNADPH ratio of 0.15.The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.
l‐α‐Hydroxyacid Oxidase Isozymes
Authors: DULEY John A. , HOLMES Roger S. 
Published: March 2005

l‐α‐Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 ± 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000.The total amino acid contents of the two rat isozymes were very similar and had identical tyrosine (9 residues per 40000) and tryptophan (5 per 40000) contents. No free suiphydryl groups were found following suiphydryl titration experiments using the purified native rat isozymes. Following denaturation with sodium dodecylsulphate, 2 (A subunit) and 3 (B subunit) suiphydryl residues were titratable. The apoenzyme form of rat isozyme B exhibited 2 of the 3 titratable suiphydryl groups in the absence of sodium dodecylsulphate which suggests an involvement of these residues in coenzyme binding. Flavin mononucleotide content was estimated from spectral studies to be I residue per subunit (40000 molecular weight) of native enzyme. Following storage in ammonium sulphate at 4°C for several months, flavin mononucleotide was irreversibly removed such that the rat A and B isozymes contained 0.3 and 0.7 residue of coenzyme per subunit. This accounts for the more pronounced loss of specific activity following storage for the A isozyme than the B isozyme as well as the comparatively high minimal molecular weights based upon flavin mononucleotide values previously reported by other workers.This evidence for chemical homology together with the previously reported immunochemical crossreactivity for these isozymes provide evidence that the A and B polypeptides are products of recently duplicated genes during evolution.
Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872
Authors: GRIFFIN Martin , TRUDGILL Peter W. 
Published: March 2005

Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40‐fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis.
The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three or four subunits of molecular weight 54000–58000.
The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.
The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixedfunction oxygenases with external electron donors. The oxygenated product has been identified as 1‐oxa‐2‐oxocyclohexane (5‐valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2‐lactonizing) (EC 1.14.13.–).
Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p‐hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent.
The enzyme has a pH optimum of 7.7. The Km for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits.
Anaerobic reduction of enzyme‐bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments.
Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated.
Temperature‐Dependent Change in Rate‐Limiting Step of the Magnesium‐Stimulated ITPase of Myosin
Authors: HOZUMI Tetsu 
Published: March 2005

The effects of temperature on Mg‐ITPase activity of heavy meromyosin and myosin sub‐fragment 1 were measured in 0.1 M KCl. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 °C. However, it was almost zero mol below 8 °C. Effects of KCl concentration and pH on ITPase activity of heavy meromyosin at 20 °C were different from those below 8 °C, suggesting that the rate‐limiting step in the Mg‐ITP hydrolysis of myosin depends on temperature.The effect of temperature on the actin activation of heavy meromyosin Mg‐ITPase was analyzed by measuring the temperature dependence of double‐reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at lower temperature.The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.