bluuebunny/crossref_metadata_2025 huggingface dataset:
Statistical Estimations in Enzyme Kinetics
Authors: Fernley Herman N.
Published: March 2005

A procedure for fitting experimental data directly to the integrated Michaelis equation is described. It involves choosing parameters that minimize the sum of the squares of deviations due to errors in s, the substrate concentration at time t. Estimates of the standard errors of the parameters are provided.
The Occurrence of Multiple Activities in the High‐Molecular‐Weight DNA Polymerase Fraction of Mammalian Tissues
Authors: Holmes Andrew M. , Hesslewood Ian P. , Johnston Irving R.
Published: March 2005

The high‐molecular‐weight DNA polymerase fraction of calf thymus, rat liver or rat spleen can be resolved into further activities differing from one another in molecular weight and charge properties. The use of Sepharose 6B columns brings about partial resolution of these activities although DEAE‐cellulose achieves a more effective separation. The high‐molecular‐weight fraction from calf thymus has so far yielded four species of activity, A, B, C and D sedimenting at 7.9 S, 5.2 S, 7.3 S and 6.0 S, respectively. In rat liver and spleen, 7.6‐S (I), 7.23 (II) and 5‐S enzymes have been detected although smaller and more variable amounts of 5‐S enzyme occur in the rat tissues compared with calf thymus where the 5.2‐5 species can represent 20–25%, of the total activity. There is no evidence for the presence of the 3.4‐S enzyme in the partially purified fractions.

All enzymes exhibit anomalous gel filtration behaviour; it is suggested that this is due to asymmetry of the molecules. The molecular weights for both calf and rat enzymes are drawn from the ranges 200000–225000, 155000–170000 and 100000–110000; since these are multiples of 50000–55000, a main band on dodecylsulphate‐polyacrylamide gel electrophoresis of molecular weight 53000–54000 for one calf enzyme and one rat enzyme, is of special interest.

Of the four calf thymus activities only enzyme D can utilise poly(dA) · (dT)10 to support [3H]dTMP incorporation. The pooled peaks of A, B and C from calf thymus all utilise poly(dT) · (A)10 to support [3H]dAMP incorporation. A, B and C of calf thymus have pH optima in the region of 7.8–8.1 whereas D had its pH optimum at pH 7.2; Enzymes I and II of rat liver disappear together with increasing age of the animal, and like A, B, C and D of calf thymus are especially sensitive to the level of salt in the assay.
The Immunochemistry of Peptidoglycan
Authors: Schleifer Karl H. , Seidl Peter H.
Published: March 2005

A pentapeptide, Gly‐l‐Ala‐l‐Ala‐d‐Ala‐d‐Ala, was synthesized and covalently linked with its free amino group to human serum albumin. Immunization of rabbits with this albumin‐peptide conjugate yielded antisera containing predominantly antibodies directed against the peptide. By means of inhibition studies with various amino‐aicd derivatives and related peptides, it was demonstrated that the antibodies are directed against the C‐terminal portion of the pentapeptide and that d‐alanyl‐d‐alanine is the immunodominant site of the antigen. These antibodies showed also a specific cross‐reaction with solubilized peptidoglycan containing peptide subunits with C‐terminal d‐alanyl‐d‐alanine and with another albumin‐peptide conjugate carrying Gly‐γ‐d‐Glu‐l‐Ala‐d‐Ala‐d‐Ala pentapeptide residues. These albumin‐peptide conjugates also reacted with rabbit antisera of Streptococcus group‐A‐variant which contain antibodies directed against the peptide moiety of the peptidoglycan. Double‐gel diffusion studies with antiserum to Streptococcus group‐A‐variant or to albumin‐peptide conjugate as antibody source and albumin‐peptide‐conjugate and a soluble high molecular fraction of peptidoglycan as antigens yielded precipitin lines which completely fused. This indicates that these antigens have identical determinant groups with respect to the antisera employed.
Human Pancreatic Proteolytic Enzymes and Protein Inhibitors
Authors: Feinstein Gad , Hofstein Ronny , Koifmann Joseph , Sokolovsky Mordechai
Published: March 2005

The purification of human proteolytic enzymes, i.e. two trypsins, two chymotrypsins and at least one elastase from activated extracts of human pancreas has been accomplished by a combination of affinity chromatography and conventional ion‐exchange chromatography on SE‐Sephadex. The enzymes thus obtained were homogeneous by several criteria: gel electrophoresis, equilibrium sedimentation and immunological assay. The molecular weight and amino‐acid composition of each preparation was determined and compared with those of other mammalian enzymes. The two trypsins showed only partial immunological identity while the two chymotrypsins were identical immunologically. The data raises the question of the number of the zymogens present in the human pancreas.

In addition to the proteolytic enzymes, four protein inhibitors of proteolytic enzymes were also isolated from human pancreas. The procedure consisted of several steps that included gel filtration, affinity chromatography and isoelectric focusing. The two major components which accounted for most of the trypsin inhibitory activity were characterized and were apparently of the Kazal type. The more basic human pancreatic inhibitor had a pI of 8.7 and contained 48 amino acids with a molecular weight of 5200. The more acidic inhibitor had a pI of 6.5 and contained 39 amino acids with a molecular weight of 4300. Both inhibitors were capable of inhibiting bovine and two human trypsins. They did not inhibit two human chymotrypsins but had a weak capacity to inhibit bovine chymotrypsin.
The Specificity of the Rat‐Liver Cholesterol 7α‐Hydroxylase
Authors: Brown Maureen J. G. , Boyd George S.
Published: March 2005

Experiments have been performed which investigate the relationship of the cholesterol 7α‐hydroxylase enzyme to the cytochrome‐P ‐450‐dependent mixed‐function oxidase system of rat‐liver microsomes. There appears to be no simple relationship between the activity of the cholesterol 7α‐hydroxylase enzyme and the cytochrome P ‐450 content of rat liver microsomes. Rat liver microsomal cholesterol 7α‐hydroxylase was sensitive to cycloheximide treatment. Its activity decayed in vivo following this treatment more rapidly than the aminopyrine demethylase enzyme or the mixed‐function oxidase components cytochrome P ‐450 and NADPH‐cytochrome c reductase. Freezing and thawing of rat liver microsomes resulted in a marked decrease in the activity of the cholesterol 7α‐hydroxylase.

The specificity of the cholesterol 7α‐hydroxylase enzyme has been investigated by comparing the liver microsomal oxidation of cholesterol with that of other steroids having a similar structure to cholesterol.

The substrates whose oxidation was studied were androst‐5‐en‐3β‐ol‐17‐one, pregn‐5‐en‐3β ol‐20‐one, androst‐5‐en‐3β‐ol and pregn‐5‐en‐3β‐ol. All these steroids were metabolised to one or more products when incubated with rat liver microsomes, NADPH and oxygen. Treatment of rats with cholestyramine which increased the activity of cholesterol 7α hydroxylase, had no effect on the rate of oxidation of any of the four steroids studied.

The conversion of [4‐14C]cholesterol to 7α‐hydroxy[4‐14]cholesterol was significantly increased by preincubating the added tracer cholesterol with liver microsomes prior to starting the hydroxylation reaction with NADPH.

It is suggested that the observed differences between the mechanism of control of the cholesterol 7α‐hydroxylase enzyme and other mixed‐function oxidase enzymes in rat liver microsomes might be due to presence of different rate‐limiting steps in the overall enzyme complex.
Reductive‐Methylation Studies on L7 and LI2 Ribosomal Proteins
Authors: Amons Reinout , Möller Wim
Published: March 2005

L7 and L12 ribosomal proteins were reductively methylated in isolation and in situ, i.e. bound to the 50‐S ribosomal subunit. The peptide bonds involving the carboxyl groups of ε‐N‐methylated lysines are resistant to trypsin attack.

Comparison shows that most of the lysine residues of L7 and L12 proteins are more reactive in situ than in isolation. A partial unfolding or reduced association in situ is proposed. Notably when modified in solution, the reactivity of the acetylated form (L7) was greater than that of the deacetylated form (L12).

After reductive methylation the L7 and L12 proteins remain active in the GTPase reaction dependent on elongation factor G.
On the Specificity of Carbohydrate‐Lectin Recognition
Authors: Podder Sunil K. , Surolia Avadhesha , Bachhawat Bimal K.
Published: March 2005

A galactose‐specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature‐dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively.

The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A‐specific sugars. The complex formation results in a time‐dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time‐dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein‐protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell‐lectin interaction is discussed.
6‐O‐Methyl‐d‐glucosamine in Lipopolysaccharides of Rhodopseudomonas palustris Strains
Authors: Mayer Hubert , Framberg Karola , Weckesser Jürgen
Published: March 2005

A hitherto unknown amino sugar was discovered in lipopolysaccharides isolated from strains of chemotype III of the photosynthetic bacterium Rhodopseudomonas palustris. The sugar (2–3% of the lipopolysaccharides dry weight) is detected as a shoulder on the descending part of the glucosamine peak obtained when acid hydrolysates of the lipopolysaccharides are passed through an amino acid analyzer. It was separated from the other amino sugars of the lipopolysaccharide (i.e. glucosamine, galactosamine, quinovosamine) by high‐voltage paper electrophoresis and subsequent paper chromatography. The mass spectrometric analysis of the isolated sugar as deuterium‐reduced alditol acetate revealed the presence of a 6‐O‐methyl‐2‐amino‐2‐deoxyhexose. Demethylation of the isolated sugar afforded glucosamine and the demethylation of its ninhydrin degradation product yielded arabinose, which showed that the parental amino sugar is glucosamine. Authentic 6‐O‐methyl‐d‐glucosamine exhibited identical properties in paper chromatography and high‐voltage electrophoresis as did its alditol acetate derivative in gas‐liquid chromatography and mass spectrometry. These data prove that the unknown amino sugar in lipopolysaccharides of Rhodopseudomonas palustris is 6‐O‐methyl‐glucosamine. The strong positive optical rotation of the isolated sugar indicates its d‐configuration.

6‐O‐Methyl‐d‐glucosamine is part of the O‐specific chain and not of thelipid A moiety of the lipopolysaccharide. Periodate‐oxidation studies revealed that the sugar, like the other amino sugars of this lipopolysaccharide, does not occupy non‐reducing terminal positions to any appreciable extent.
Electron‐Paramagnetic‐Resonance Study of Manganese Ions Bound to Concanavalin A
Authors: Von Goldammer Eberhard , Zorn Herbert
Published: March 2005

The electron paramagnetic resonance (EPR) line shape of Mn2+ ions bound in crystallized concanavalin A has been analyzed at two microwave frequencies, 9 and 35 GHz. From a comparison between measured and simulated EPR‐spectra, the correlation times for the process modulating the zero field splitting (zfs) interaction and the magntiude of the zfs‐parameters have been determined. In the absence of Ca2+ ions a correlation time of 10 ps (at 4°C), typical for the thermal motions of the diffusing transition metal ions, has been determined. In the presence of Ca2+ ions the Mn2+‐spectrum of concanavalin A reveals forbidden transitions (Δm=±1), indicating that the mean life‐time for the transition metal ions at the binding site S1 is increased (τass≳ 10 ns) under the action of calcium ions bound at S2.
Proton‐Magnetic‐Resonance Studies of Complexes of Pancreatic Ribonuclease A with Pyrimidine and Purine Nucleotides
Authors: Haar Wolfgang , Rüterjans Heinz , Maurer Wolfgang
Published: March 2005

In continuation of earlier work new results on the behaviour of certain proton resonances of pancreatic ribonuclease A and of nucleotides during complex formation are reported. By varying the structure of the base or the position of the phosphate group at the ribose moiety it can be shown that the main structural feature of the ribonuclease · pyrimidine‐nucleotide complexes is maintained. However, the microscopic binding constants of the interactions of the various parts of a nucleotide with the corresponding amino acid sidegroups are different for nearly all the investigated complexes. While the modified purine base of 8‐oxoadenosine 3′‐phosphate seems to be bound in the pyrimidine binding site of ribonuclease A also, the base of the other adenosine nucleotides studied occupy other sites at the enzyme. For adenosine 3′‐ and 5′‐monophosphate this might be the binding site shown to be specific for purine bases by X‐ray crystallography. But the results obtained for the ribonuclease · adenosine‐2′‐phosphate complex suggest that the adenosine nucleotides do not bind exclusively in this purine binding site.
Specific Inactivation of Fructose 1,6‐Bisphosphatase from Saccharomyces cerevisiae by a Yeast Protease
Authors: Molano Jesús , Gancedo Carlos
Published: March 2005

A fraction has been isolated from baker's yeast that is able to inactivate fructose 1,6‐bisphos‐phatase in vitro. The inactivating fraction is heat labile, non‐dialysable and is precipitated with ammonium sulfate; thus indicating that it is a protein. Malate dehydrogenase, hexokinase, glucose phosphate isomerase, glucose‐6‐phosphate dehydrogenase, glutamate dehydrogenase and catalase from baker's yeast and fructose 1,6‐bisphosphatase from the yeast Rhodotorula glutinis were not inactivated by the preparation.

A fraction which is able to inhibit the inactivating factor has also been found in baker's yeast. The inhibitor is thermoresistant, non‐dialysable and precipitable by ammonium sulfate. The possible biological significance of these findings in relation with the regulation of yeast fructose 1,6‐bisphosphatase is considered.
The Hexose‐Proton Symport System of Chlorella vulgaris
Authors: Komor Ewald , Tanner Widmar
Published: March 2005

The specificity of the hexose transport system of Chlorella for sugars is the same as the specificity of sugar‐induced proton uptake. The Km‐values of various sugars for the rate of proton uptake agree well with the respective Km‐values for the rate of uptake of these sugars.

Under anaerobic conditions in the dark, when the rate of sugar uptake is very low, sugar‐stimulated proton uptake is also hardly observable. Under the same conditions in the light [712 nm; O2 evolution is inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea] sugar again stimulates proton uptake. The uncoupler carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone, which inhibits sugar uptake, also prevents sugar induced proton uptake.

The Stoichiometry for 6‐deoxyglucose and for glucose under various energetic conditions is one proton taken up per sugar molecule. For other glucose analogues the Stoichiometry has been found to be larger than one. Potassium and sodium ions cannot replace protons in the hexoseproton symport system.
The Poly(adenylic acid) Sequence of Mouse Globin Messenger RNA
Authors: Mansbridge Jonathan N. , Crossley Jennifer A. , Lanyon W. George , Williamson Robert
Published: March 2005

Mouse globin messenger RNA has been degraded by ribonuclease T1 to give poly(adenylic acid) fragments of two size classes which run electrophoretically with standard RNA molecules approximately 75 and 50 nucleotides long. These contain no internal ribonucleotides other than adenosine and occur at the 3′‐end of the mRNA molecule. The probable sequence to the 5′‐side of the poly(A) tract is ‐G‐C. Attempts to separate the α and β mRNA activities by formamide gradient elution from poly(U)‐Sepharose were unsuccessful.

The evidence is more compatible with a post‐transcriptional end‐addition mechanism for the synthesis of the poly(adenylic acid) region of messenger RNA than a transcriptional mechanism.
Lack of Incorporation of 1: N6‐Ethenoadenosine Triphosphate into Yeast tRNAPhe and tRNASer under Standard Conditions
Authors: Zachau Hans G. , Hertz Harry S.
Published: March 2005

The previously reported incorporation of the 1: N6‐ethenoadenosine monophosphate (ɛAMP) moiety of ɛATP into baker's yeast tRNAphe and tRNASer is an artifact which arose from the presence of small amounts of ATP in the ɛATP preparations. Sensitive assays for ATP contamination in ɛATP are described.
On the Mechanism and Control of the Malonyl‐CoA‐Dependent Chain Elongation of Fatty Acids
Authors: Podack Eckhard R. , Lakomek Max , Saathoff Gisela , Seubert Werner
Published: March 2005

The purification of the microsomal fatty acid chain elongating system from beef adrenal cortex is described.

The elongation system requires for optimal activity malonyl‐CoA, ATP, Mg2+, NADPH or NADH. Long‐chain fatty acyl‐CoA derivatives inhibit the incorporation of [1,3‐14C]malonyl‐CoA into fatty acids. Supplementation of the system with enzymes of the C‐3 oxidation sequence allows also an acetyl‐CoA‐dependent chain elongation.

Microsomes from beef adrenal cortex contain 2,3‐trans‐hexenoyl‐CoA reductase with similar properties as described for rat liver.

The ratio of the specific activities of the hexenoyl‐CoA reductase and chain elongation remains constant in the course of purification from rat liver and beef adrenal cortex. From this finding it is concluded that hexenoyl‐CoA reductase is an integrated part of the microsomal malonyl‐CoA‐dependent chain elongation system.

The physiological substrates of hexenoyl‐CoA reductase are 2,3‐unsaturated acyl‐CoA derivatives of polyenoic long‐chain fatty acids with a preference for structures present in γ‐linolenic acid.

Competitive inhibition of the microsomal enoyl‐CoA reductase by malonyl‐CoA with respect to enoyl‐CoA suggests that a multienzyme complex is involved in the microsomal chain elongation process.

Octenoyl‐CoA, octenoyl‐pantethein and octenyl‐acyl‐carrier protein are substrates of the microsomal hexenoyl‐CoA reductase from rat liver with decreasing maximal velocities and affinities. Octenoyl‐acyl‐carrier protein and malonyl‐CoA are competitive, suggesting identical mechanisms of reduction for octenoyl‐acyl‐carrier protein and octenoyl‐CoA by the multienzyme complex.
Intrinsic Luminescence Studies on the Apoenzyme and Holoenzyme of Lipoamide Dehydrogenase
Authors: Visser Antonie J. W. G. , Grande Hans J. , Müller Franz , Veeger Cees
Published: March 2005

The following fluorescence parameters have been investigated: emission and excitation spectra, relative quantum yields, polarization values and lifetimes of the apo‐ and holoenzyme of lipoamide dehydrogenase. Furthermore, fluorescence perturbation techniques have been used to characterize qualitatively the tryptophan and flavin environment in the protein. Also emission spectra at low temperature have been recorded to examine changes occurring on strong immobilization of the enzyme. From lifetimes and quantum yields of the protein fluorescence it can be concluded that the molecular structures of the two apoenzymes, which can be prepared from this enzyme, are different. The quenched protein fluorescence of the holoenzyme is characterized by a very short fluorescence lifetime due to radiationless energy transfer from tryptophan to flavin. Excitation polarization spectra of flavin and tryptophan fluorescences show that the chromophores are rigidly bound to the protein. Luminescence studies at liquid nitrogen temperatures reveal that the triplet state of the tryptophanyl‐residues is not involved in energy transfer to the flavin.

Collisional fluorescence quenching with KI shows in the dimeric holoenzyme, in contrast with the free molecules, only a small reduction in fluorescence intensity of tryptophanyl and flavin residues. Charge effects might be responsible for these observations. It can be derived from the experimental results together with the indication that only one of the two tryptophans is transferring its excitation energy to the flavin, that the distance between donor and acceptor is 1.3–1.6 nm.
Release of Glucose from Purified Galactose‐Binding Protein of Escherichia coli upon Addition of Galactose
Authors: Richarme Gilbert , Kepes Adam
Published: March 2005

The galactose‐binding protein which forms an essential component of the galactose transport system, called β‐methyl galactose permease, shows a galactose binding behavior that cannot be described by the usual association‐dissociation equilibrium equations. It was found that the amount of [14C]galactose bound was dependent on the ratio of the amounts of galactose to galactose‐binding protein present in the reaction mixture. It is shown furthermore, that addition of galactose to its binding protein results in the release of another ligand, which by an enzymatic assay and by thin‐layer chromatography was shown to be glucose. The glucose liberated upon addition of [14C]galactose was non‐radioactive, and approximately equivalent to the [14C]galactose bound. An exchange equilibrium between glucose and galactose can account for the distorted “binding” kinetics.
Gross Conformation of Human Secretory Immunoglobulin A and Its Component Parts
Authors: Björk Ingemar , Lindh Erik
Published: March 2005

Human secretory immunoglobulin A, several immunoglobulin A dimers and monomers and secretory component have been characterized with regard to size, shape and conformation. The molecular weights obtained were 420000 for secretory IgA, 360000 for IgA dimer, 160000 for IgA monomer and 75000 for secretory component. Secretory IgA has a markedly extended shape as evident from its frictional ratio and intrinsic viscosity. Moreover, the hydrodynamic parameters of secretory IgA are similar to those of dimeric serum IgA, which indicates similar molecular shapes of the two proteins and thus that the presence of the secretory component does not have a significant influence on the shape of the IgA part of the secretory IgA molecule. This is in contrast to results of electron‐microscopic investigations, which have suggested different shapes for secretory IgA and IgA dimer. The hydrodynamic data are compatible with an extended model for both proteins, in which the carboxy‐terminal halves of the IgA mononiers are joined end to end via a rigid or flexible link, but are in less agreement with an alternative, more compact model, in which the two IgA monomers are superimposed on each other. The IgA monomer itself has hydrodynamic characteristics suggesting a significantly more extended shape than that of IgG. Optical rotatory dispersion and circular dichroism spectra show large similarities between the conformations of IgG and both secretory and serum IgA, although minor differences do exist. As in IgG, evidence for the presence of β‐structure in the different types of IgA was obtained. The similarities of the ORD and CD spectra of secretory IgA and IgA dimer suggest that the secretory component does not exert a major effect on the internal folding of the IgA backbone.

Human secretory component also has a somewhat extended shape. Its conformation, as measured by ORD and CD spectra, differs markedly from that of the immunoglobulins, although the spectra do seem to suggest the presence of some β‐structure also in secretory component. The main ORD minimum and a prominent CD band exhibited by this protein are very rarely seen in proteins and suggest that a unique type of conformation is present in secretory component.
Interactions between Membrane Functions and Protein Synthesis in Reticulocytes
Authors: Herzberg Max , Britbart Haim , Atlan Henri
Published: March 2005

In this paper we demonstrate that two compounds which are known to enhance the permeability of membranes to potassium, valinomycin, a cyclic dodecapeptide and dicyclohexyl‐18‐crow‐6, a macrocyclic polyether, inhibit protein synthesis in reticulocytes and that this inhibition is not primarily due to changes in the intracellular potassium concentration. No effect is obtained by adding the drugs to a cell free system and the inhibition observed is likely to be membrane mediated.

Dicyclohexyl‐18‐crown‐6 and low concentration of valinomycin (10 nM) act reversibly probably by interacting with the lipid phase of the membrane. This interaction in turn affects the protein synthesizing apparatus by affecting ribosomal functions. Ribosomes once isolated from treated cells are perfectly normal in structural and functional respects.

High concentrations of valinomycin, which change the K+ gradient between the inside and the outside of the cell, affect the protein synthesizing apparatus of reticulocytes irreversibly by blocking incorporation of amino acids at the level of elongation or termination. Ribosomes isolated from cells treated with high valinomycin concentrations are mostly in the form of polysomes which are inactive in cell free protein synthesis.

The high valinomycin block on the ribosomal fraction can be relieved either by washing ribosomes with high salt or with puromycin. It is believed that with high concentrations of valinomycin an inhibitor is released (probably from the membrane) and attached to the ribosomes. This high valinomycin effect is believed to by a supplementary phenomenon which when added to the effect of low valinomycin renders the inactivation of ribosomal functions irreversible.
On the Mechanism of Glucose‐6‐Phosphate Dehydrogenase Regulation in Mouse Liver
Authors: Yagil Gad , Shimron Frida , Hizi Amnon
Published: March 2005

The activity of glucose‐6‐phosphate dehydrogenase in mouse liver is induced nearly 10‐fold and the activity of citrate lyase nearly 6‐fold, when C57BL mice are transferred from a diet containing 25% corn oil to a fatless diet. When the mice on the fatless diet are transferred back to the high‐fat diet, both enzymes return to their basal level with half‐lives of 41 and 55 h, respectively. Malate‐NADP dehydrogenase and 6‐phosphogluconate dehydrogenase are induced and repressed in parallel, but to a lesser extent. In addition to the induction‐repression cycle, all four enzymes also undergo diurnal variations involving 50%, changes in their activities. Glucose‐6‐phosphate dehydrogenase is induced to a certain extent also in adipose tissue, but not in several other mouse tissues. The induction cycle can be repeated in liver at least three times successively. Glucose‐6‐phosphate dehydrogenase induction takes place in the bulk of parenchymal liver cells and not in a selected population. Actinomycin D and cycloheximide administration prevents the induction of the enzymes, but it is not clear whether a direct effect on the enzyme‐synthesizing machinery is involved.
Binding of Secretory Component to Dimers of Immunoglobulin A in vitro
Authors: Lindh Erik , Björk Ingemar
Published: March 2005

The binding of human free secretory component to four different dimeric myeloma immunoglobulins A has been studied in vitro. Two of the immunoglobulin dimer preparations bound equimolar amounts of secretory component, while the other two showed lower binding capacity. The 1:1 molar complexes were investigated further. A certain number of antigenic determinants of secretory component were buried upon complex formation, while no corresponding blocking of immunoglobin A determinants could be detected. The molecular weights of the complexes (about 420000) agreed well with that of naturally occurring secretory immunoglobulin A, as did their shape and conformation, the latter as studied by optical rotatory dispersion and circular dichroism. Thus the binding of secretory component to dimeric immunoglobulin A in vitro can be used as a model to elucidate the interactions between the two moieties in secretory immunoglobulin A. These interactions appear to have both a noncovalent and a covalent component. Only minimal conformational changes accompany complex formation, indicating that the noncovalent interactions between the two proteins involve no major structural rearrangements of either molecule.
Kinetics of Pyrophosphate‐ATP Exchange Catalysed by l‐Tryptophan:tRNA Ligase from Escherichia coli
Authors: Penzer Geoffrey R. , Plumbridge Jacqueline A.
Published: March 2005

A kinetic analysis of the pyrophosphate‐ATP exchange reaction catalysed by l‐tryptophan :tRNA ligase purified from Escherichia coli K‐12 EM‐20031 has been performed. It is based on rate measurements at 80 sets of substrate concentrations. The result indicates that the enzyme binds both ATP and l‐tryptophan (Kd‐values are 1030 and 15 μM, respectively) but not pyrophosphate. Binding of ATP and l‐tryptophan together is synergistic, the Kd‐values being decreased by two thirds. Comparison of the results with those obtained for other amino acid: tRNA ligases shows that (a) dissociation constants vary widely and (b) synergistic binding has rarely been detected for true substrates, though it is well documented for a variety of substrate analogues.
Myeloperoxidase of Human Neutrophilic Granulocytes as Chlorinating Enzyme
Authors: Stelmaszyńsky Teresa , Zgliczyński Jan M.
Published: March 2005

Two pH‐dependent spectral forms of myeloperoxidase have been described. Their absorption maxima in the Soret region were at 432 nm and 426 nm for acid and neutral pH, respectively. Chloride exerts an influence on the spectrum of peroxidase, mainly in acidic medium. Dissociation constants for the myeloperoxidase chloride complex and Km values for chloride in the chlorination reaction catalyzed by myeloperoxidase were determined. It was found that the affinity of chloride ions to the enzyme decreases with an increase of pH.

Catalytic activity of myeloperoxidase in the reaction of incorporation of 36Cl into aliphatic amino compounds such as taurine, β‐alanine, ethanolamine and diethanolamine was investigated. Primary amino groups were chlorinated to N‐mono‐ or dichloramines, depending on the pH of the medium. The method of determination of 36Cl‐labelled chlorocompounds in the presence of excess Na36Cl was described.

The role of myeloperoxidase as chlorinating enzyme in the mechanism of the bactericidal effect on bacteria phagocytized by neutrophilic granulocytes is discussed.
The Sequence of Sheep κ‐Casein: Primary Structure of para‐κA‐Casein
Authors: JOLLÈS Jacqueline , SCHOENTGEN Françoise , HERMANN Jacques , JOLLÈS Pierre , ALAIS Charles
Published: March 2005

The primary structure of sheep para‐xA‐casein, the N‐terminal portion of x‐casein, was established. The reduced alkylated protein was subjected to digestion with trypsin. The resulting soluble peptides were purified by a combination of Dowex 1 × 2 chromatography and electrophoresis and chromatography on paper; the core peptides were solubilized in 50% formic acid and filtered on Sephadex G‐50. The amino‐acid sequence of these peptides was determined chiefly with a Sequencer. Alignment of the tryptic peptides into a single chain containing 105 amino acids was determined from basic overlap peptides (tryptic core peptides with several basic amino acids; chymotryptic peptides). Some comparisons were achieved with cow para‐xA‐Casein.
Competition for ADP between Pyruvate Kinase and Mitochondrial Oxidative Phosphorylation as a Control Mechanism in Glycolysis
Authors: GOSALVEZ Mario , PÉREZ‐GARCÍA Jesús , WEINHOUSE Sidney
Published: March 2005

To assess a possible role of pyruvate kinase as a site for respiratory and glycolytic interaction, competition for ADP between pyruvate kinase and respiratory phosphorylation was measured in a model system consisting of rat liver mitochondria respiring in the presence of pyruvate kinase, phosphoenolpyruvate, ATP, and an ADP‐regenerating system consisting of glucose and purified yeast hexokinase. This system allowed determination of total ATP production, equivalent to ADP utilization, by measuring glucose 6‐phosphate formation; ADP utilization by pyruvate kinase by pyruvate formation; and respiratory ADP utilization by Pi uptake. O2 uptake was measured by means of an oxygen electrode. In the presence of a respiratory substrate such as succinate or glutamate‐malate, the addition of pyruvate kinase and phosphoenolpyruvate reduced O2 uptake as well as oxidative phosphorylation about 80%. Respiration was increasingly inhibited with increasing pyruvate kinase, and this inhibition was decreased with increased hexokinase or ATP. Mitochondrial respiratory inhibition by pyruvate kinase and phosphoenolpyruvate was accompanied by an increase in the ATP/ADP ratio from 0.3 to 32. These inhibitory effects of pyruvate kinase on respiration were abolished by addition of 2,4‐dinitrophenol. These results are in acccord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with oxidative phosphorylation for the available ADP; and provide additional support for our previous suggestion that high glycolysis of certain tumors may be attributable to their extraordinarily high pyruvate kinase activity.
Reassociation and Reactivation of Lactic Dehydrogenase from the Unfolded Subunits
Authors: JAENICKE Rainer
Published: March 2005

Lactic dehydrogenase from pig heart (H4) shows pH‐dependent reversible deactivation, denaturation and dissociation at pH < 6.

The initial and final states in the dissociation‐association process are found to be the homogeneous fully inactive monomer (Mr= 37500 ± 1000) on one hand, and a heterogeneous mixture of denatured subunits, native tetramers and inactive higher aggregates on the other hand. The yield of tetramers and the extent to which renaturation occurs correspond to each other.

Using optimum conditions of reactivation [pH 7.9, I∼ 0.2, 20 °C, in the presence of 1 mM dithiothreitol and 10 mM EDTA, enzyme concentration below 0.05 mg/ml], the correlation of reactivation and reassociation may be analyzed. The second‐order kinetics of the reactivation process prove reassociation to be the rate‐limiting step in the process of reactivation. From this one may conclude that in the case of the heart isoenzyme of lactic dehydrogenase the subunit does not represent the enzymatically active unit.

The coenzyme (NAD+) increases the rate of reactivation but not the overall recovery. This suggests the coenzyme is involved in a nucleation step in the process of refolding and/or reassociation.
On the First Steps of Lactate Oxidation by Bakers' Yeast L‐(+)‐Lactate Dehydrogenase (Cytochrome b2
Authors: LEDERER Florence
Published: March 2005

The behaviour of bakers' yeast l‐lactate dehydrogenase toward [2‐2H]lactate 3‐chlorolactate and 2‐hydroxy‐3‐butynoate is reported. By comparison with known facts concerning d‐amino acid and lactic oxidases, the following conclusions are drawn.

1. Hydrogen removal at C‐2 is rate‐limiting in the overall reaction. This distinguishes l‐lactate dehydrogenase from the oxidases where it is product dissociation which limits the rate.

2. The lack of dehydrohalogenation of 3‐chlorolactate by l‐lactate dehydrogenase, which differentiates the enzyme from the oxidases, can be explained if the reduction rate of flavin is much faster than for the oxidases.

3. Although only indirect evidence is presented, it seems most probable that the lactate C‐2 hydrogen is removed as a proton, as in the case of d‐amino acid oxidase and lactic oxidase.

4. The first steps of lactate oxidation are qualitatively similar for the two known enzymatically active forms of l‐lactate dehydrogenase.
The Role of the Sulphydryl Groups of Spleen Deoxycytidylate Aminohydrolase
Authors: CERVONE Felice , VACCARO Carlo , SEPE Santo , SCARANO Eduardo , ROSSI Mosé
Published: March 2005

The relation between sulphydryl groups and enzyme activity, and between sulphydryl groups and changes of conformation of the enzyme molecule caused by allosteric effectors has been studied in deoxycytidylate aminohydrolase with a homogeneous preparation of the spleen enzyme. Binding of 4 moles of p‐chloromercuribenzoate per mole is sufficient to inactivate the enzyme. Three different time rates of inactivation by p‐chloromercuribenzoate are obtained for the enzyme, for the enzyme · dTTP‐Mg complex and for the enzyme · dCTP‐Mg complex. The 4 thiol groups on the enzyme responsible for the inactivation by p‐chloromercuribenzoate are not available to the action of 5,5′‐dithio‐bis(2‐nitrobenzoic acid). Experiments of titration of the enzyme –SH groups by 5,5′‐dithio‐bis(2‐nitrobenzoic acid) give additional support to the contention that at least three conformers are involved in the regulation of the activity of deoxycytidylate aminohydrolase. The data suggest that –SH groups have a very important role in catalysis and regulation of the enzyme.
The Determination of the Primary Structure of Histone F3 from Chicken Erythrocytes by Automatic Edman Degradation
Authors: BRANDT Wolf F. , VON HOLT Claus
Published: March 2005

In order to determine the sequence of histone F3 by automatic Edman degradation, this protein was fragmented into a series of smaller fragments. Specific chemical cleavages rather than enzymatic degradations were applied in such a way as to yield a limited number of larger fragments.

In the first step the histone F3 dimer was cleaved at the two methionine residues with cyanogen bromide. The three fragments were separated and purified by gel filtration. Their respective sizes were 90, 30 and 15 amino‐acid residues.

The two larger fragments were both cleaved with N‐bromosuccinimide at tyrosine residues. They in turn yielded three and two fragments, respectively, which could be readily purified by gel filtration and carboxymethyl‐cellulose ion‐exchange chromatography.

One of the larger fragments obtained by cleaving the 90 residue fragment at its tyrosine residues was further degraded by dilute acid into another three fragments and two free aspartic acid residues which also could be purified by gel filtration.

The position of all the 10 fragments in the protein molecule became apparent by comparing the N‐ and C‐terminal amino‐acid residues of the starting material and the cleaved products after each cleavage.

The simplicity of the peptide mixture after each cleavage resulted in an easy separation of the fragments.
Membrane‐Bound Transpeptidase and Penicillin Binding Sites in Streptomyces Strain R61
Authors: MARQUET Alberto , DUSART Jean , GHUYSEN Jean‐Marie , PERKINS Harold R.
Published: March 2005

High‐affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4°C in 0.017 M K2HPO4 or 0.05 M Tris‐HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [14C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [14C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane‐bound transpeptidase. The same rate constant (about 1 × 10−4 s−1) for the dissociation of the benzylpenicillin membrane complex at 37°C and in 0.017 M K2HPO4, was calculated either from the release of the radioactivity (using [14C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high‐affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37°C and in 0.017 M K2HPO4 at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10−3 to 0.73 × 10−4 s−1. The radioactivity released through the dissociation of [14C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [W]‐benzylpenicilloic acid both by paper electrophoresis and thin‐layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule.
Facilitation by Adenosine of the Action of Insulin on the Accumulation of Adenosine 3′:5′‐Monophosphate, Lipolysis, and Glucose Oxidation in Isolated Fat Cells
Authors: SCHWABE Ulrich , SCHÖNHÖFER Peter S. , EBERT Reinhold
Published: March 2005

The modulation of insulin action by adenosine has been studied in lipid and glucose metabolism of isolated rat fat cells. When 20000 cells per ml were incubated, insulin failed to antagonize the effect of 1 μM noradrenaline on adenosine 3′:5′‐monophosphate (cyclic AMP) levels and lipolysis. At high concentrations (100000 cells per ml) both parameters were strongly depressed by insulin. The addition of 0.1 μM adenosine to dilute cell suspensions decreased cyclic AMP accumulation in response to noradrenaline, but only marginally reduced lipolysis, thus producing the same situation as seen in concentrated cell suspensions. Furthermore, insulin antagonized the cyclic AMP accumulation and lipolysis caused by adenosine deaminase (1 μg per ml) or theophylline (0.3 mM). These two agents only moderately elevated cyclic AMP levels similar to the effect of noradrenaline in the presence of adenosine. These data strongly indicate that insulin is able to counteract hormone effects on cyclic AMP formation and lipolysis only at low cellular levels of the cyclic nucleotide.

Stimulation of glucose oxidation by insulin in the range between 10 and 100 μU per ml was markedly enhanced in the presence of 0.1 μM adenosine when only 5000 fat cells per ml were incubated. On the other hand, adenosine deaminase reduced the action of insulin in the range between 1 and 300 μU per ml in concentrated cell suspensions (100000 cells per ml). The uptake of adenosine and the ATP levels in fat cells were not altered by insulin, except at high concentrations of adenosine (0.1 mM). Theophylline (0.5 mM) and dipyridamole (20μM) abolished the stimulatory effect of both insulin and adenosine on glucose oxidation. Increasing concentrations of adenosine were able to overcome the inhibitory effect of theophylline but not that of dipyridamole. These data suggest that adenosine may have an essential regulatory role which can be described as facilitation of insulin actions, even though different mechanisms apply to lipolysis and glucose oxidation.
On the Mechanism of Ribonuclease A
Authors: SAENGER W. , SUCK D. , ECKSTEIN F.
Published: March 2005

The triethylammonium salt of uridine 3′‐O‐thiophosphate methyl ester crystallized in space group P21 with cell dimensions a= 684.5 pm, b= 1480.4 pm, c= 2254.2 pm, β= 92.8° and two molecules per asymmetric unit. Solution of the structure was accomplished by direct methods; the nucleotide molecules have been located unambiguously but the cations are disordered and appear only as highly noise background. The least‐squares refinement converged at a conventional R‐factor of 19.1%.

The base in both molecules of the asymmetric unit is anti with respect to the sugar, the conformation about the C(4′)‐C(5′) bond is gauche, gauche, the sugar pucker is C(2′)‐endo. The conformation about the two P–O ester bonds is not as observed in double helical RNA's, suggesting that the sulphur atom directs the orientation of the ester bonds.

Uridine 3′‐O‐thiophosphate methyl ester was obtained by pancreatic ribonuclease A catalyzed methanolysis of the endo‐isomer of uridine 2′: 3′‐cyclothiophosphate. As the thiophosphate diester moiety is in the R configuration with respect to the methyl group, the enzyme must have attacked the cyclothiophosphate via an in‐line mechanism.
Hormonal Mechanisms Regulating Hepatic Steroid Metabolizing Activities
Authors: ERIKSSON Håkan
Published: March 2005

The possible influence of androgens and estrogens on the sexual differentiation of hepatic enzymes metabolizing corticosterone was studied in the isolated perfused rat liver.

The predominant metabolites in livers from normal male rats were 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one, 5α‐pregnane‐3α,11β,20α, 21‐tetrol and 5α‐pregnane‐3β,11β,20β,21‐tetrol excreted as mono‐ and disulphates.

Castration of adult male rats resulted in a reduced formation of 5α‐pregnane‐3,11β,20,21‐tetrols and a decreased ratio between 20β and 20α epimers. The amount of sulphate conjuged metabolites was increased.

Partial hepatectomy of castrated adult male rats followed by complete regeneration of the liver parenchyma resulted in formation of 3α,11β,2 1‐trihydroxy‐5α‐pregnan‐20‐one.

Administration of 100μg estradiol benzoate to normal adult male rats slightly reduced the amounts of 5αa‐pregnane‐3,11β,20,21‐tetrol epimers formed, whereas the same injection to castrated adult male rats markedly decreased the 20α (and 20β) oxidoreductase activity.

The most pronounced effect of estradiol benzoate administration was seen in castrated adult male rats subjected to partial hepatectomy followed by complete parenchymal regeneration under estradiol influence. In these rats the metabolite profile was almost the same as that seen after perfusion of female rat livers with corticosterone. The predominant metabolite was 3αa,11β,15α,21‐tetrahydroxy‐5α,14ξ‐pregnan‐20‐one, which has never been found in the male rat. The activities of the 3‐ and 20‐hydroxysteroid oxidoreductases were almost the same as those found in livers from normal female rats. Furthermore, a major part of the metabolites were monosulphates which is typical of the female rat.

The present data show that estrogens are capable of shifting conjugation and oxidoreductase activities in liver cells previously exposed to androgens towards a female pattern. However, an estrogen‐mediated induction of a 15‐hydroxylase active on corticosterone, which is a typical reaction in the female rat liver, can only take place in liver cells which have not previously been exposed to androgens.
Mössbauer Studies of Some Complexes of the Undecapeptide of Cytochrome c
Authors: BAUMGARTNER Celia P. , SELLERS Martha , NASSIF Rosemarie , MAY Leopold
Published: March 2005

Complexes of cytochrome c and of an undecapeptide of the protein have been studied by means of Mössbauer spectroscopy in order to obtain information concerning the active heme site.

The variation of the quadrupole splitting with the temperature permits an evaluation of the distortions from octahedral symmetry around the iron ion in the heme group, and also an estimation of the spin‐orbit constant. The effect of the different ligands such as CN−, F−, imidazol, etc., is discussed.
Enzymes of Nitrogen Metabolism in Legume Nodules
Authors: BOLAND Michael J. , BENNY A. Graeme
Published: March 2005

An NADH‐dependent glutamate synthase has been purified 500‐fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2‐oxoglutarate, glutamine and NADH are 39 μM, 400 μM and 1.3 μM respectively. The catalytic centre activity is of the order of 70 s‐1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid. aspartic acid and asparagine, all competitive with 2‐oxoglutarate; and by NAD+, which is competitive with NADH.

There is evidence of two flavine prosthetic groups per enzyme molecule.