bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Subunit Structure of Rabbit‐Skeletal‐Muscle Phosphorylase Kinase, and the Molecular Basis of Its Activation Reactions
Authors: Cohen Philip 
Published: March 2005

Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion‐exchange chromatography, and antigen‐antibody precipitation in agar. The purified enzyme showed four protein‐staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units.Activation of the enzyme by incubation with adenosine‐3′:5′‐phosphate‐dependent protein kinase, adenosine 3′:5′‐phosphate and Mg‐ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.
Inhibition of Protein Biosynthesis in Escherichia coli B by Tri‐l‐ornithine
Authors: Barak Zeev , Sarid Sara , Katchalski Ephraim 
Published: March 2005

Triornithine completely inhibited growth of Escherichia coli B in the minimal liquid medium M63. The di‐, tetra‐ and pentapeptides showed no effect on the growth of the bacterium.
Triornithine, when added to a bacterial culture, inhibited the incorporation of 14C‐labeled amino acids into the protein fraction, but did not suppress the incorporation of [3H]uridine and [3H]thymidine.
The tripeptide inhibited translation of the induced β‐galactosidase mRNA and also inhibited the synthesis of alkaline phosphatase initiated by derepression.
Triornithine had no effect on the reaction in vitro of T4‐DNA‐directed RNA synthesis.
Incubation of E. coli B cells with triornithine impaired the protein biosynthetic system of the treated cells in vitro. By this treatment the biological activity of the ribosomal fraction is impaired within the bacterial cell.
Triornithine, even at a high concentration, did not inhibit in vitro the polypeptide synthesis in a cell‐free system.
Inhibition of T4 Maturation by Tri‐l‐ornithine
Authors: Barak Zeev , Sarid Sara , Katchalski Ephraim 
Published: March 2005

Triornithine inhibited the development of new T4D phages within the bacteria. The plaque‐forming capability of free phages was not affected.
Triornithine inhibits the synthesis of early, as well as late protein in T4‐infected Escherichia coli B cells.
Triornithine was found to enhance [3H]uridine incorporation into viral mRNA. [3H] Thymidine incorporation into T4 DNA was not affected by the tripeptide when added after the time interval during which all of the enzymes concerned with viral DNA synthesis have already been formed.
Two Forms of Microsomal Palmitoyl‐Coenzyme A Synthetase
Authors: Brandes Ruth , Zohar Yizhak , Arad Rivka , Shapiro Benyamin 
Published: March 2005

Two palmitoyl‐CoA synthetase activities have been demonstrated in rat liver microsomes. One, catalyzing palmitoyl‐CoA synthesis from ATP, palmitate and CoASH as well as from palmitoyl‐AMP and CoASH. A second enzyme reacts with the first constituents only and is inactive with palmitoyl‐AMP. The first enzyme, but not the second catalyzes the reaction between palmitoyl‐AMP and PPi to yield ATP and palmitic acid.This duality has been shown by: (a) partial separation of the two types of activity by fractional solution in Triton X‐100, (b) kinetic behaviour testing competition between both reaction mixtures at saturating concentrations and (c) the varying distribution of the two activities in microsomes obtained from rats under different dietary regimes or when diabetes was induced by alloxan.
Structure of Protocollagen Proline Hydroxylase from Chick Embryos
Authors: Olsen Bjørn R. , Berg Richard A. , Kivirikko Kari I. , Prockop Darwin J. 
Published: March 2005

Protocollagen proline hydroxylase was purified from chick embryos with an affinity‐column procedure which yielded pure enzyme in a tetramer form. Electron microscopy of the enzyme after it was largely dissociated into monomers indicated that the monomers were rod‐shaped with a diameter of 3.31 ± 0.07 nm (S.E.M.) and a length of 6.95 ± 0.07 nm. In preparations of the enzyme in which the protein was partially dissociated, dumb‐bell and V‐shaped structures were seen which morphologically consisted of two sub‐structures each with dimensions similar to those of the isolated monomers. The results suggested that these structures were dimers in which the monomers were joined at one end with an angle between their longitudinal axes.The largest regular protein structures seen in preparations of the enzyme were about 8 × 8 nm and in some of these structures four sub‐structures were seen, indicating that they were tetramers. On the basis of the data presented here we propose a model of the tetramer in which two V‐shaped dimers were interlocked.Further studies demonstrated that the single‐ring and four‐ring structures seen by electron microscopy of enzyme preparations obtained by a previous purification procedure were not the enzyme.
Characteristics of Nuclear‐Ribosomal and DNA‐Like Ribonucleic Acids Differentially Extracted by Hot‐Phenol Fractionation
Authors: Markov George G. , Arion Vitali J. 
Published: March 2005

The purpose of the present study was to define more exactly the conditions of the method of hot‐phenol fractionation of nuclear RNA (Georgiev and associates). The thermal RNA fractions obtained were characterized by agar‐gel electrophoresis, determination of the specific radioactivity and base composition analysis, both of the total and newly synthesized RNA.It was found that the fraction extracted at 0–4 °C (considered as cytoplasmic RNA in the literature) contained some nuclear ribosomal precursor RNAs, expecially 32‐S and 23‐S RNAs.The composition of the different thermal RNA fractions strongly depended on the extraction conditions (time, pH, volume ratiosetc.). Conditions were well‐defined (15‐min extraction at each temperature, pH 6.2) for obtaining, as separate fractions, the nuclear RNA species of ribosomal type (40‐°C RNA,i.e.that obtained at 40 °C) and two classes of DNA‐like RNA (63‐°C and 85‐°C RNA). The 55‐°C RNA fraction was found to be of mixed character (rRNA + DNA‐like RNA) regardless of the extraction conditions.The 85‐°C RNA contained the “giant” RNA molecules, had the highest specific radioactivity and a (G + C)/(A + U) ratio of about 0.80. These findings are in agreement with the view that 85‐°C RNA is the very first product of transcription. 63‐°C RNA was also DNA‐like but different from 85‐°C RNA with respect to electrophoretic profile, specific radioactivity and base composition. A high content of adenylic acid (above 30%) was found in the region 8 to 18 S of 63‐°C RNA. It is suggested that poly(A) segments are attached at the 18‐S stage of the processing of 63‐°C RNA as a precursor to the cytoplasmic mRNA.The results presented demonstrate that under well‐defined conditions the method of the hot‐phenol fractionation of Georgiev may be used as a reproducible procedure for obtaining different types of nuclear RNA without preliminary isolation of nuclei and nucleoli.
The Time Course of Recombination of Human Chorionic Gonadotropin Subunits Studied with Immunological Methods, Circular‐Dichroic Measurements and Bioassay
Authors: Merz Wolfgang E. , Hilgenfeldt Ulrich , Brockerhoff Peter , Brossmer Reinhard 
Published: March 2005

The restitution of immunological properties of native human chorionic gonadotropin by recombination of the subunits, α and β, was studied by immunological methods (qualitative and two‐dimensional immunoelectrophoresis and two immunodiffusion methods). Recombination of the subunits at different incubation times was carried out with three different mixture ratios. The bulk of the recombined hormone is formed during the first hour. The immunological behaviour of recombined hormone corresponds largely to that of the starting material used for the preparation of the subunits. In addition to the recombined hormone material a further recombination product appears which can be detected neither in the subunit preparations nor in the native hormone. It seems to be formed in high concentrations immediately after the beginning of incubation and to decrease rapidly thereafter. With an excess of β subunit in the recombination mixture, however, it does not disappear completely. The second product possesses antigenic determinants different from those of the recombined hormone. The circular dichroic spectra of the recombination mixtures display variations in dichroic absorption during the course of recombination, as well as a third maximum. With increasing incubation time, the third maximum decreases and is shifted to the far ultraviolet region, where it finally overlaps with the extremum at 195 nm.The biological activity of the reconstituted material reaches as much as 75% of the starting material.
Murex trunculus Haemocyanin
Authors: Wood Edward J. , Peacocke Arthur R. 
Published: March 2005

The native or whole molecule of the haemocyanin from the whelk, Murex trunculus had a sedimentation coefficient of 102.7 S, and combination of this with the diffusion coefficient of 1.03 × 10−7 cm2 s−1 led to a molecular weight of 8.85 × 106. The parameter β of Scheraga and Mandelkern was 2.14 × 106, and the frictional ratio f/f0 was 1.5 indicating that the molecule did not deviate markedly from the spherical. Electron microscopy showed that the molecule was a short cylinder, about 35 nm in diameter.When the pH was raised dissociation occurred into halves and then into smaller subunits which were referred to as “twentieths”, but which were heterogeneous. This dissociation took place at pH values greater than 7.6 in the absence of Mg2+, or at pH values near 10.0 in presence of 0.01 M MgCl2. Whole molecules were re‐formed from halves upon lowering the pH, but “twentieths”, once formed, did not re‐associate. The products of dissociation were separable by preparative ultracentrifugation and by zone electrophoresis. The proportion of wholes, halves, and twentieths formed was determined by the pH and Mg2+ concentration and was not influenced by rotor speed or by protein concentration. It was considered that there was not a dynamic equilibrium between wholes, halves and twentieths.In 6 M guanidine‐HCl in the presence of 2‐mercaptoethanol a single species of molecular weight about 220000 was formed. In detergent‐gel electrophoresis there was rather poor evidence for a component of molecular weight about 50000, which whould correspond with one oxygen‐binding site containing 2 copper atoms.
ω‐Hydroxylation of Steroid Side‐Chain in Biosynthesis of Bile Acids
Authors: BjÖRKHEM Ingemar , Gustafsson Jan 
Published: March 2005

ω‐Hydroxylation (26‐hydroxylation) of various C27‐steroids by the microsomal and mitochondrial fraction was studied. Assay conditions for the microsomal 26‐hydroxylation were determined with 5β‐cholestane‐3α,7α‐diol as substrate. NADPH was the required cofactor. NADH stimulated the hydroxylation at suboptimal concentrations of NADPH. The hydroxylation was inhibited markedly by carbon monoxide. Treatment with phenobarbital inhibited the reaction whereas starvation or biliary drainage had no significant effect. The rates of microsomal 26‐hydroxylation of different C27‐steroids expressed in nmol/mg protein per 20 min were cholesterol, <0.1; 5‐cholestene‐3β,7α‐diol, <0.1; 7α‐hydroxy‐4‐cholesten‐3‐one, 0.3; 7α,12α‐dihydroxy‐4‐cholesten‐3‐one, 0.9; 5β‐cholestane‐3α,7α‐diol, 1.8; 5β‐cholestane‐3α,7α, 12α‐triol, 2.7.Assay conditions for the mitochondrial 26‐hydroxylation were determined with cholesterol and 5β‐cholestane‐3α,7α‐diol as substrates. The rate of reaction was faster with an NADPH‐generating system than with NADPH. The reaction was inhibited markedly with carbon monoxide. Biliary drainage inhibited the reaction whereas starvation or treatment with phenobarbital had no significant effect. The rates of mitochondrial 26‐hydroxylation of different C27‐steroids expressed in nmol/mg protein per 20 min were: cholesterol, 0.3; 5‐cholestene‐3β,7α‐diol, 0.9; 7α‐hydroxy‐4‐cholesten‐3‐one, 1.7; 7α,12α‐dihydroxy‐4‐cholesten‐3‐one, 0.9; 5β‐cholestane‐3α,7α‐diol, 0.9; 5β‐cholestane‐3α,7α,12α‐triol, 2.0.The results are consistent with previously postulated pathways concerning biosynthesis of cholic acid and chenodeoxycholic acid in which 5β‐cholestane‐3α,7α,12α‐triol and 5β‐cholestane‐3α,7α‐diol, respectively are the main substrates for the 26‐hydroxylase. The possibility is discussed that pathways involving intermediary formation of 7α,26‐dihydroxy‐4‐cholesten‐3‐one and 7α,12α,26‐trihydroxy‐4‐cholesten‐3‐one also are of importance in the biosynthesis of bile acids.
A Transport System for 2‐Keto‐3‐deoxy‐d‐gluconate Uptake in Escherichia coli K12
Authors: Lagarde Alain E. , Pouysségur Jacques M. , Stoeber François R. 
Published: March 2005

We have presented kinetic evidence for the occurrence in Escherichia coli K 12 of an active transport system responsible for 2‐keto‐3‐deoxy‐d‐gluconate uptake. It has been studied in constitutive strains PK4 and CAK 101 since it is completely cryptic in the wild‐type strain.

The accumulation is temperature‐dependent, coupled to electron and energy‐yielding reactions, and is inhibited by thiol reagents.
2‐Keto‐3‐deoxy‐d‐gluconate is not transported through the phospho‐enolpyruvate‐transferase system.
Entry reaction displays saturation kinetics obeying the Michaelis‐Menten laws. The Km value has been found to be 0.25 ± 0.05 mM (at 30 °C and pH 7.0), by measuring initial rates of entrance as well as steady‐state levels. Both the operator‐constitutive PK4 strain and the regulator‐constitutive CAK101 strain exhibit the same affinity but apparent V are 10 and 20 nmol × mg‐1× min‐1, respectively.
The 2‐keto‐3‐deoxy‐d‐gluconate permeation system is endogenously induced in a strain lacking 2‐keto‐3‐deoxy‐d‐gluconate kinase, when grown on any intermediary product of the hexuronide catabolic pathway, able to enter the cell and anterior to 2‐keto‐3‐deoxy‐d‐gluconate in the sequence. We presented further evidence making it very likely that 2‐keto‐3‐deoxy‐d‐gluconate itself is the true inducer.
The constitutive synthesis of the transport system is sensitive to the catabolite repression exerted by glucose and pyruvate.
By testing a wide number of sugars and derivatives as possible substrates, only d‐glucuronate is a strictly competitive inhibitor (Ki= 1.5 mM). Since a specific hexuronate permease has been described elsewhere, we were led to demonstrate that d‐glucuronate could be taken up through both its own specific system with high affinity and through the 2‐keto‐3‐deoxy‐d‐gluconate specific system with lower affinity.
The exit reaction rate is first oder with respect to internal concentration, temperature‐dependent (exit rate constant kex is 0.0180 min‐1 at 30 °C) but is not carrier‐mediated.
The Flux of Pyruvate in Perfused Rat Heart
Authors: Mowbray John , Ottaway James H. 
Published: March 2005

It was possible to find a steady‐state lactate concentration in a closed‐circuit heart perfusion containing added glucose. Insulin but not growth hormone (4 μg/ml) increased the equilibrium concentration in perfusate by 200%. Intracellular lactate also remained constant and was about twice that in perfusate: this distribution is difficult to explain whether ionic or undissociated lactate is the permeating species.
Pyruvate accumulated linearly in perfusate, reaching a peak after 30 min perfusion and then declined non‐linearly. Insulin increased the peak concentration and growth hormone slowed the decline after the peak. The intracellular pyruvate concentration stayed constant and, except in the first few minutes, was lower than the perfusate concentration.
Pyruvate addition at zero time could inhibit pyruvate accumulation but altered neither the non‐linear decline, the intracellular pyruvate level nor the lactate equilibrium.
The [NAD]/[NADH] ratio, after 10 min of pre‐perfusion on open circuit, was 23. During 2 h of perfusion the ratio rose slowly and then declined. If either insulin or growth hormone were present in the medium, the ratio fell to about 11 during the first few minutes of perfusion and remained low for 90 min. These values could not easily be correlated with either the varying extracellular, or the constant intracellular [lactate]/[pyruvate] ratios.
The glucose and oxygen consumption of the hearts during perfusion was measured, and the change in glycogen concentration in the tissue. It was then possible to draw up a balance sheet which indicated that the control hearts oxidised little other than carbohydrate for the 60 min of observation, but both insulin and growth hormone‐treated hearts oxidised significant quantities of fatty acid during the first 15 min. It appears that growth hormone, like insulin, has an immediate effect on heart metabolism. The addition of pyruvate at zero time appeared to stimulate carbohydrate oxidation in hearts perfused with insulin, even though the hormone had been present in the pre‐perfusion fluid.
Consideration of the variation in the extracellular [lactate]/[pyruvate] ratio with length of perfusion, and its lack of relationship to the intracellular [lactate]/[pyruvate] ratio, together with similar reports of other workers, leads us to conclude that neither lactate nor pyruvate establish a concentration gradient across the heart cell membrane which is based on passive diffusion. The discrepancy is particularly marked with respect to pyruvate. Our results strongly suggest that the extracellular [lactate]/[pyruvate] ratio cannot be used as an index of the cytoplasmic redox potential in perfused heart.
A tracer study of pyruvate metabolism in the reproducible non‐steady‐state conditions described can offer an explanation for the behaviour of the observed metabolic parameters: this study is reported in the following papers.
Identification of the Lipid Intermediate in Yeast Mannan Biosynthesis
Authors: Jung Peter , Tanner Widmar 
Published: March 2005

The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP‐mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane‐bound enzymes. The lipid component has been purified more than 1000‐fold (based on lipid‐bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate.Yeast dolichol is a mixture of five homologous polyprenols with saturated α‐isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.
Pyruvate Kinase
Authors: Carbonell Juan , Marco Roberto , Felíu Juan E. , Sols Alberto 
Published: March 2005

Kinetic studies of the pyruvate kinases of rat and human tissues have led to the identification of three classes of isoenzymes with qualitative differences in regulatory properties.Class L, the major component in liver extracts and a minor component of kidney extracts, shows markedly sigmoidal kinetics with respect to the concentration of phosphoenolpyruvate, allosteric inhibition by ATP and alanine, and activation by fructose 1,6‐bisphosphate. Class A, present in adipose tissue, and the major component in kidney and the minor one in liver, shows slightly sigmoidal substrate saturation curves and is allosterically inhibited by alanine and activated by fructose 1,6‐bisphosphate. Class M, present in muscle and brain, has none of the above regulatory properties.The activities of the pyruvate kinases of classes L and A respond immediately to changes in the concentration of the effectors alanine and fructose 1,6‐bisphosphate.The two regulatory isoenzymes are strongly inhibited by three amino acids: alanine, cysteine, and phenylalanine. Cysteine is the stronger inhibitor for class L, while phenylalanine is the stronger one for class A. The allosteric inhibition is stereospecific for the l‐amino acids in contrast with the weak isosteric inhibition of muscle pyruvate kinase by l‐ or d‐alanine as analogue of the product.The allosteric inhibition of pyruvate kinase L by ATP is highly specific in contrast with a rather wide specificity of this enzyme for nucleoside diphosphates as acceptor substrates.
Photosystem‐II Electron Transport and Phosphorylation with Dibromothymoquinone as the Electron Acceptor
Authors: Gould J. Michael , Izawa Seikichi 
Published: March 2005

Dibromothymoquinone has two effects on isolated chloroplasts. At very low concentrations it inhibits the reduction of conventional hydrophilic electron acceptors, probably by acting as a plastoquinone antagonist. At higher concentrations it acts as an electron acceptor, intercepting electrons either before or at the site of its inhibitory activity. Reduced dibromothymoquinone can be readily reoxidized by excess ferricyanide in the reaction mixture or by molecular oxygen. The transfer of electrons to this substance from water is coupled to phosphorylation. The pH optima for this reduction and associated phosphorylation are both at 7.3, considerably lower than the pH optimum of 8.4 observed with the normal Hill reaction. The ratio of molecules of ATP formed to pairs of electrons transported is relatively constant (0.3 –0.4) between pH 6 and 9. The rate of reduction is independent of the presence or absence of ADP and phosphate or uncouplers.The reduction of dibromothymoquinone resembles the reduction of other lipid‐soluble oxi‐dants such as oxidized p‐phenylenediamines and 2,5‐dimethylquinone in several respects. Both reactions are insensitive to the plastocyanin inhibitors KCN and polylysine, but are sensitive to 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. They support phosphorylation with a similar efficieney. However, dibromothymoquinone reduction differs from the reduction of other lipophilic oxidants in that its sensitivity to dichlorophenyl‐dimethylurea decreases with increasing light intensities. This implies that it may be reduced via a pool (plastoquinone?) which is a common electron acceptor for independent photosystem II units whereas oxidized p‐phenylenediamines and 2,5‐dimethylquinone are reduced directly by these independent units.