bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Aspects of Heterologous Blood‐Group‐P1 Substance
Authors: Anstee David J. , Pardoe Grace I.
Published: March 2005

Blood‐group‐P1 substance from sheep hydatid‐cyst fluid was purified by a combination of gel filtration through Sepharose 6B and preparative polyacrylamide‐gel electrophoresis. The resultant glycoprotein contained D‐galactose; D‐glucose; N‐acetyl‐D‐glucosamine and N‐acetyl‐D‐galactosamine in molar ratios 5.2:1:2:2; unidentified carbohydrate was also present. The amino‐acid composition resembled that of vertebrate A, B, H and Lewis active glycoproteins.

Prolonged incubation of P1 substance with coffee bean α‐galactosidase resulted in the liberation of 17–24% of the total D‐galactose present and a reduction in blood‐group‐P1 inhibitory activity. The serological activity of the glycoprotein was unaffected by treatment with pronase or papain.

P1 substance has receptors for the agglutinins from Salmo trutta, Clupea harengus, Soja hispida, Maclura aurantiaca and Triticum vulgaris. Some loss of receptor for Soja hispida and Maclura aurantiaca occurred after α‐D‐galactosidase treatment.
Interconvertible Forms of Phosphofructokinase of Rabbit Liver
Authors: Ramaiah Abburi , Tejwani Gopi A.
Published: March 2005

When the phosphofructokinase reaction was started with the addition of stock rabbit liver phosphofructokinase its activity declined rapidly, at low concentrations of fructose 6‐phosphate or at high concentrations of ATP. The rate of this decline is independent of enzyme concentration and it could be prevented by the presence of positive effectors of the enzyme or by high concentration of fructose 6‐phosphate.

When the reaction rate was measured in the first few seconds after addition of stock enzyme, the activity of the enzyme and its [S]0.5 value (concentration of fructose 6‐phosphate for half maximal velocity) were not affected significantly by the presence of positive effectors of the enzyme.

The stock enzyme, which was routinely stored in the presence of 0.7 M (NH4)2SO4, 10 mM K2HPO4, 10 mM ATP, 50 mM Tris‐HCl pH 7.5 and 0.33 mM EDTA, was transformed to a less active form of enzyme, which had a very high [S]0.5 value and low intrinsic activity on dilution or when freed of effectors by passing the enzyme solution through a Sephadex G‐25 column. This form can be converted back to the active form by the positive effectors of the enzyme. The rate of this conversion of less active form to the active form of enzymes is again independent of enzyme concentration.

The rate‐limiting step in the interconversions of active form to less active form of enzyme or vice versa is a first‐order process and there appears to be no aggregation or disaggregation of the enzyme during these interconversions.

A variety of [S]0.5 values and maximal velocities were observed for the enzyme depending on the preincubation, prior dilution and removal of the effectors of enzyme suggesting the existence of at least two conformational states of enzyme in varying proportions differing widely in their [S]0.5 values and their intrinsic activities.

These results are interpreted in terms of the model of Monod et al. for allosteric enzymes with minor modifications and the hysteretic concept of Frieden.
The Active Hexose‐Uptake System of Chlorella vulgaris
Authors: Komor Ewald , Haass Dieter , Komor Brunhild , Tanner Widmar
Published: March 2005

The Km value for 6‐deoxyglucose influx into Chlorella vulgaris was found to be 0.21 mM and for efflux it was 21 mM. The sugar was accumulated up to 1500‐fold, an accumulation which cannot be explained by the difference in Km values.

Using these Km values together with estimated relative translocation constants for carrier and carrier‐substrate it was possible to determine Ks values. The Ks value thus obtained for the carrier at the inner side of the membrane was 70 times larger than that on the outer side.

Although different Km values to the ones given above for influx as well as for efflux were obtained when the measurements were carried out in the presence of high sugar concentrations at the opposite side of the membrane, the calculations yielded almost identical Ks values (Table 3).

The experimentally observed accumulation of 6‐deoxyglucose can be well explained when the different Ks values and the estimated difference of 25‐fold between the translocation constants for outward as compared to inward “movement” of free carrier is considered.
Transport of Hexoses in Streptomyces violaceoruber
Authors: Sabater Bartolomé , Asensio Carlos
Published: March 2005

Transport of glucose, mannose and fructose has been studied in spores and mycelia of Streptomyces violaceroruber grown under a variety of nutritional conditions.

Each of the three sugars is transported into mycelial cells by a specific, constitutive transport system. The apparent Michaelis constants of these systems for glucose, mannose and fructose are 1, 1.5 and 3 mM, respectively. At least in the case of mannose and fructose, transport seems to be of the non‐active type.

In contrast with vegetative cells, spores of Streptomyces appreciably transport only glucose, with a Km of 1 mM, but not mannose and fructose. This observation can be related to the lag for growth of Streptomyces spores on mannose or fructose with respect to growth on glucose.
Tryptophanyl‐Transfer Ribonucleic‐Acid Synthetase from Beef Pancreas
Authors: Iborra François , Dorizzi Mireille , Labouesse Julie
Published: March 2005

Binding of tryptophan, tryptamine and ATP to tryptophanyl‐tRNA synthetase was studied by equilibrium dialysis experiments. There are two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could be measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits.

Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi‐ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active dimer into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer‐monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species.

The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state.
Evolution of Ribosomal Proteins
Authors: Delaunay Jean , Creusot Francine , Schapira Georges
Published: March 2005

We have studied the evolution of ribosomal proteins over a spectrum of 13 species ranging from man to plants using a two‐dimensional polyacrylamide gel electrophoresis technique. We tried to quantify the proximity of these species by coelectrophoresing the proteins from various combinations of two species. The following tentative conclusions have been reached.

For reptiles and birds which diverged 300 million years ago from the line leading to mammals, ribosomal proteins display the same fingerprints as mammals.

For amphibians and fish which diverged 350–400 and 440–500 million years ago respectively, the fingerprints show limited differences compared with mammals. Those of fish show some diversity within the species.

For lower species (crustaceans, molluscs) and plants, the fingerprints exhibit a great diversity.
Pantetheine‐Free Mutants of the Yeast Fatty‐Acid‐Synthetase Complex
Authors: Schweizer Eckhart , Kniep Bernhard , Castorph Helga , Holzner Ulrich
Published: March 2005

Among 52 Saccharomyces cerevisiae fatty acid synthetase (fas)mutants screened for their ability to incorporate 14C‐labeled pantothenic acid into the fatty acid synthetase multienzyme complex, especially those of fas‐complementation group VII had lost this ability. The purified fatty acid synthetase complexes of all the 19 fas‐mutants available from this group were shown to be free of 14C‐labeled pantothenic acid. The amount of pantothenate incorporated into the enzymes of several other fas‐mutants of the non‐pleiotropic complementation groups II, V, VI and VIII, however, was similar to that observed with wild‐type fatty acid synthetase. The purified pantothenate‐free fatty acid synthetases of five different group VII fas‐mutants have been tested for the seven known component enzymes of the complex. In all mutants, only the β‐ketoacyl synthetase was inactive whereas in vitro all the other partial activities were unimpaired. By sodium dodecylsulfate‐polyacrylamide‐gel electrophoresis, no differences could be observed between the protein structure of the pantothenate‐free mutant fatty acid synthetase and that of the wild‐type comp ex. Both were separated into three different components A, B and C with molecular weights of 185000, 180000 and 177000, respectively. However, some fas‐mutants consist only of the components A and B, others only of B and C. The study of various [14C]‐pantothenate‐labeled mutant fatty acid synthetases suggests that component C originates from A presumably by limited proteolysis. In the undegraded complex AB, [14C]pantothenate is only associated with the component A. Since in one of the mutants studied A is completely converted to C, it is concluded that A is one distinct component rather than a group of components with identical molecular weights. It is tentatively suggested that the gene product of fas 2, one of the two known fatty‐acid‐synthetase gene clusters in yeast, is only one, single and multifunctional polypeptide chain. Therefore, it appears that in yeast, the acyl carrier protein is not an individual protein component of the fatty acid synthetase complex, but only a distinct region of the multifunctional polypeptide chain encoded by fas 2.
The Role of Histidine Residues in Yeast Hexokinase
Authors: Grouselle Michel , Thiam Abdul Aziz , Pudles Julio
Published: March 2005

Specific acylation of the histidine residues of yeast hexokinase with diethylpyrocarbonate at pH 6.1 or 7.5 leads to a partial and reversible inactivation of the enzyme.

The carboethoxylation of the histidine residues at pH 7.5 was faster than at 6.1 and the loss in activity was also higher.

However the difference in the amount of inactivation was related to a greater instability of the carboethoxylated enzyme at pH 7.5.

When the carboethoxylation was carried out at pH 7.5 and at 0 °C, only the histidines residues were modified (nine residues per enzyme subunit) and still 40% of the activity was left.

d‐Glucose protects the enzyme activity in the course of the carboethoxylation at pH 7.5, but has no effect at pH 6.1. However the rate and number of histidines acylated were the same as when the experiments were carried in absence of the substrate.

All these results clearly indicate that the histidine residues are not implicated either in the catalytic or in the substrate binding site of yeast hexokinase.

This was also confirmed by showing that the 60% inactivated enzyme had the same Km as the native enzyme for both substrates d‐glucose and Mg · ATP. Only v was affected.

The inactivation effect of the carboethoxylation was not related to a displacement of the pH optimum of the activity, since the enzymes species having five and eight histidine residues modified‐respectively in absence and presence of d‐glucose at pH 7.5 gave the same pH profile of activity as the native enzyme.

Absorption spectroscopy, fluorescence and ultracentrifugation studies have shown that the loss of enzymic activity in the course of the modification of the histidine residues is only related to changes of the protein conformation.
A Small‐Angle X‐Ray Scattering Study on Poly[d(A‐T) · d(A‐T)], Poly[d(A‐s4T) · d(A‐s4T)] and Poly[d(A‐s4U) · d(A‐s4U)]
Authors: Zipper Peter
Published: March 2005

A small‐angle X‐ray scattering study was performed with solutions of the alternating polydeoxynucleotide poly[d(A‐T) · d(A‐T)] and its analogs poly[d(A‐s4T) · d(A‐s4T)] and poly[d(A‐s4U) · d(A‐s4U)]. At low concentration of NaCl and neutral pH almost identical results were obtained for these polydeoxynucleotides. At small scattering angles the same cross section radius of gyration Rc= 0.92 ± 0.03 nm for all three polydeoxynucleotides was derived from the scattering curves. At larger scattering angles a second cross‐section radius of gyration Rc2 could be determined which amounts to 0.855 ± 0.02 nm for poly[d(A‐T) · d(A‐T)], 0.86 ± 0.01 nm for poly‐[d(A‐s4T) · d(A‐s4T)] and 0.82 ± 0.01 nm for poly[d(A‐s4U) · d(A‐s4U)], respectively. The length per nucleotide pair, 0.33 ± 0.03 nm, was found to be the same for all three polydeoxynucleotides. These results are similar to those for natural DNA.

The above results for poly[d(A‐s4T) · d(A‐s4T)] and poly[d(A‐s4U) · d(A‐s4U)] refer to the modification of these polydeoxynucleotides which prevails at low concentrations of 1:1 electrolytes and was designated as helix I. The length per nucleotide pair obtained in this study appears to be consistent with the presence of strong 0 0 8 and 0 0 16 reflections in the high‐humidity X‐ray fibre diffraction patterns for the sodium salt of poy[d(A‐s4T) · d(A‐s4T)]. The values for the cross‐section radii of gyration, on the other hand, seem to be inconsistent with the reversed Hoogsteen base‐pairing scheme proposed for poly[d(A‐s4T) · d(A‐s4T)] and poly[d(A‐s4U) · d(A‐s4U)], because the similarity of these values to those for poly[d(A‐T) · d(A‐T)] and natural DNA is contrary to the results of theoretical calculations on models built up from reversed Hoogsteen base‐pairs. According to these calculations, the cross‐section radius of gyration of poly‐[d(A‐s4T) · d(A‐s4T)] ought to be smaller than that of natural DNA by about 0.16–0.2 nm if this polydeoxynucleotide really were of the reversed Hoogsteen type.
Purification, Subunit Structure and Properties of Two Repressible Phosphohydrolases of Bacillus subtilis
Authors: Le Héagarat Jean‐Claude , Anagnostopoulos Constantine
Published: March 2005

In Bacillus subtilis 168 a class of point mutations (locus “phoP”) brings about the simultaneous loss of alkaline phosphatase and of a “phosphodiesterase” [substrate bis(p)‐nitrophenyl)‐phosphate] activities. The latter enzyme also hydrolyzes UDPG and (the phosphatase substrate) p‐nitrophenyl phosphate. In order to test a possible structural relationship between them, the two enzymes were purified and their subunit composition was studied. A new purification procedure for the alkaline phosphatase was developed; it leads to the isolation of pure enzyme with high yield. A modification of this procedure gave highly purified (but not homogeneous) preparations of “diesterase”. The molecular weights of the two enzymes are different: alkaline phosphatase 75000, “phosphodiesterase” 150000. Each enzyme is composed of two subunits of identical size; the subunits of the “diesterase” being about double in size of those of the phosphatase. The enzymes also differ in their pH optimum for p‐nitrophenyl phosphate (alkaline phosphatase pH optimum 10.2, “phosphodiesterase” pH optimum 8.0). The amino acid composition of the alkaline phosphatase was determined; the enzyme molecule is rich in basic amino acids (especially lysine). Apparent Km (for p‐nitrophenyl phosphate) (= 0.2 mM at pH 10.00) and KI (for Pi) (= 0.37 mM at pH 10.0) were measured for the purified alkaline phosphatase. Incorporation of [14C]isoleucine into proteins during Pi starvation followed by isolation of the labelled alkaline phosphatase showed that the enzyme is synthesized de novo in these conditions. The results presented indicate that (a) the enzymes are indeed repressed in presence of excess Pi and (b) they are not structurally related and, especially do not share a common subunit. The “phoP” mutations do not therefore affect a structural gene for these enzymes. Hypotheses concerning the function blocked in these mutants are discussed.
Presence of a Single Essential Histidyl Residue in Octopine Dehydrogenase as Shown by Photooxidation
Authors: Thoméa‐Beau Françoise , Olomucki Anna
Published: March 2005

Octopine dehydrogenase of Pecten maximus photooxidized in the presence of Rose Bengal was irreversibly inactivated following first‐order kinetics. The coenzyme and its analogues do not protect significantly against the photoinactivation whereas the substrates in the presence of coenzyme give an almost complete protection. About one –SH group was lost in the two cases. Moreover the native enzyme and the enzyme in which three –SH groups (including the essential one) were reversibly substituted were photoinactivated at the same rate. This suggests that the loss of the –SH group does not contribute to the photoinactivation of octopine dehydrogenase.

About two histidines were destroyed in the photoinactivated enzyme and only one in the fully protected enzyme. It seems therefore that in octopine dehydrogenase only one histidine is essential and this residue can be protected in the ternary complexes. The partial protection effects of argi‐nine or pyruvate are additive when the two substrates are used together. This fact and the lack of protective effect of the coenzyme suggest that the essential histidine residue is near the substrates binding site.
The Isolation and Properties of Two Prephospholipases A2 from Porcine Pancreas
Authors: Nieuwenhuizen Willem , Steenbergh Paul , de Haas Gerard H.
Published: March 2005

Prephospholipase A2, when isolated via the procedure described previously, by us in 1968, has pyroglutamic acid as the N‐terminal amino acid. However, this could be an artifact due to the drastic conditions of one of the early stages of the purification. Therefore a milder procedure has been developed in order to check whether or not pyroglutamic acid is artifactual. During these experiments two forms of prephospholipase A2 were purified: one (form II) being identical with the one already described, the other (form I) having a shorter activation peptide, i.e. Ser‐Ser‐Arg instead of <Glu‐Glu‐Gly‐Ile‐Ser‐Ser‐Arg.

Evidence is given that this is the only difference between forms II and I and that elastase can be responsible for the specific conversion of form II to form I by release of the N‐terminal tetra‐peptide of form II.

No carbohydrates could be detected in any of the preparations.
The Fluorimetric Determination of the Kinetic Parameters Km and kcat of the Tryptic Activation of Prephospholipase A2 and Derivatives
Authors: Nieuwenhuizen Willem , Oomens Ad , de Haas Gerard H.
Published: March 2005

The purification of two different forms of prephospholipase A2 from porcine pancreas is described in the accompanying paper. One of these has an activation peptide with the sequence <Glu‐Glu‐Gly‐Ile‐Ser‐Ser‐Arg and is designated form II; the other form has Ser‐Ser‐Arg as activation peptide and is designated form I. The Km and kcat values for the tryptic activation of forms I and II have been determined fluorimetrically, which were found to be 0.06 mM, 1.7 s−1, and 0.12 mM, 2 s−1, respectively. Chemical modifications of the free α‐amino group of form I do not affect the affinity to a great extent, but a considerable increase in kcat is observed.
Studies on the Inhibition of the L‐Serine O‐Sulphate‐Degrading System of Pig Liver and the Topography of Its Active Site
Authors: Tudball Norman , Thomas Peter
Published: March 2005

The inhibition of the l‐serine O‐sulphate‐degrading activity of pig liver has been investigated. compounds that acted as competitive inhibitors all possessed the basic stereochemistry associated with the l‐serine O‐sulphate molecule.

In general the most efficient competitive inhibitors were those possessing an interproton distance of 0.75 nm, with two electronegative ends or alternatively one electronegative end and the negative end of a dipole.

O‐Phospho‐l‐serine is an atypical inhibitor since it obeys non‐competitive inhibitor kinetics even though from theoretical considerations it should act as a competitive inhibitor.

3‐Chloro‐l‐alanine methyl ester is a substrate for the enzyme.

4‐Iodoacetamidosalicylic acid acts as an irreversible inhibitor of the system.

The probable topography of the active site and the disposition of the substrate at the active site is discussed.
Transfert du mannose, à partir de GDP‐mannose, à des accepteurs lipidiques chez Aspergillus niger
Authors: Létoublon Robert C. P. , Comte Jane , Got René
Published: March 2005

Microsomal preparations from Aspergillus niger mediate transfer of mannosyl units from GDP‐mannose to endogenous lipids. Mannosyltransferases require divalent cations for activity. Optimum pH, temperature maximal activity and optimal metal ion concentration are different whether Mn2+ or Mg2+ are used.

K
m value for GDP‐mannose is 0.2 μM in presence of Mg2+, while, with Mn2+, two values are obtained: 0.2 μM and 10 μM for low and high‐substrate concentration.

The lipid acceptor is purified by fractionation of the lipid extract on silicic‐acid column followed by thin‐layer chromatography on silica gel.

Chromatographic properties, presence of phosphorus, coloration with the anisaldehyde reagent and acid lability of the lipid suggest that this compound is a polyprenol‐phosphate.
The Substrate Specificity of Pantethinase
Authors: Duprè Silvestro , Rosei Maria Anna , Bellussi Luisa , Del Grosso Elena , Cavallini Doriano
Published: March 2005

The substrate specificty of pantethinase, the mammalian enzyme which has been isolated from horse kidney cortex, has been studied. The real substrate has been found to be pantetheine in the reduced form, and the involvement of one or more—SH groups of the enzyme is also considered.

A number of other compounds, chemically related to pantetheine, has also been tested as substrates. Pantethinase is able to metabolise pantetheine‐4′‐phosphate, with an activity about 10% of that exhibited for pantetheine; it is inactive on the other substances tested (pantothenoyl‐cysteine‐4′‐phosphate, pantothenoylcysteine, pantetheine thiazoline, CoA).

The metabolic role of pantethinase seems to be, in the light of the above results, connected with a degradative pathway of pantetheine‐4′‐phosphate, which is known to be one of the intermediates in the biosynthesis of CoA.
Purification and Properties of a Coupling Factor (Ca2+‐Dependent Adenosine Triphosphatase) from Rhodospirillum rubrum
Authors: Johansson Bo C. , Baltscheffsky Margareta , Baltscheffsky Herrick , Baccarini‐Melandri Assunta , Melandri Bruno A.
Published: March 2005

A coupling factor has been purified from an acetone powder extract of Rhodospirillum rubrum chromatophores. This restores to decoupled chromatophores (extracted with respect to coupling factor) both photophosphorylation and Mg2+‐dependent ATPase activities. The coupling factor had been partially purified by gel filtration through a Sepharose 6B column, and further purified by ultra‐filtration and sucrose‐density‐gradient centrifugation. At each stage of purification, the coupling factor activity was coincident with a Ca2+‐dependent ATPase activity, increasing in total activity during the purification. Mg2+, Mn2+, Co2+ or Zn2+ could not substitute for Ca2+ in activation of this soluble ATPase, but these four metals strongly inhibited the activity of the Ca2+‐dependent ATPase.

By incubation with decoupled chromatophores, the soluble Ca2+‐dependent ATPase was rebound to the membrane in the presence of either Mg2+ or Ca2+. This binding resulted in a partial masking of the Ca2+‐dependent ATPase activity of the soluble enzyme.

The purified ATPase has a Km value of 1.2 mM for ATP. The Ki values for inhibition of the soluble Ca2+‐dependent ATPase by Mg2+ (170 μM), and by ADP (42 μM) are in close agreement with the corresponding Km values for Mg2+‐dependent ATPase and photophosphorylation, respectively, as measured with chromatophores.

The membrane‐bound Mg2+‐ or Ca2+‐dependent ATPase activities were inhibited by oligomycin. Azide and parahydroxymercuribenzoate inhibited both the soluble and the membrane‐bound ATPase activities. Treatment with phospholipase A inhibited both the membrane‐bound ATPase, but had no effect on the soluble Ca2+‐dependent ATPase.

The sedimentation constant at 1 mg protein/ml of the extensively purified coupling factor is s20, w= 13.1 × 10−13 s, and the approximate molecular weight 350000.
Effects of Deoxyribonucleases I and II on Rat‐Liver‐Chromatin Transcription in vitro
Authors: Pays Etienne , Ronsse Annie
Published: March 2005

The hydrolysis by DNAase I or II of DNA in chromatin remains limited even after prolonged incubation.

This limitation might be due to a redistribution under the action of DNAase of f l histone

With large amounts of DNAase, all of the chromatin DNA may however be digested.

A comparison is made between the extent of DNA hydrolysis by DNAase and the resulting inhibition of chromatin template activity for endogenous, Escherichia coli or rat liver (form II) RNA polymerases. Our results are not in favour of a greater sensitivity to DNAase for the euchromatic sequences than for the repressed ones. prolonged incubation. on chromatin DNA.
Further Investigations of the Specificity of an Acid Deoxyribonuclease from Helix aspersa (Müll.)
Authors: Devillers‐Thiery Anne , Bernardi Giorgio , Ehrlich Stanislav D.
Published: March 2005

Linear relationships were found to hold between the percentages of the 3′‐P terminal and 5′‐OH terminal and penultimate nucleotides released by snail acid DNAase from bacterial DNAs and the (dG + dC) contents of the latter. This finding is similar to previous results obtained on spleen acid DNAase; as in the case of this enzyme, the composition of the termini released by the snail DNAase from calf thymus DNA devite from the linear relationships mentioned above.

The snail enzyme appears to recognize a sequence of at least two nucleotides, namely those which will be released as 3′‐P and 5′‐OH terminlas, respectively.
Une enzyme nouvelle: la 2′‐nucléotidase du latex d'Hevea brasiliensis
Authors: Jacob Jean‐Louis , Sontag Nicole
Published: March 2005

The cytoplasmic serum of Hevea brasiliensis latex contains a 2′‐nucleotidase. The monoesterase has been isolated and purified 2100 times. Its molecular weight is estimated to be about 115000–120000. Among thenucleotides, of which it hydrolyses the 2′‐phosphate group, NADP+ is utilized mainly and the nucleotides, NADPH, adenosine 2′,5′‐diphosphate, guanosine 2′,5′‐diphosphate, adenosine 2′‐phosphate, in decreasing order. In a few cases (coenzyme A, adenosine 3′,5′‐diphosphate, guanosine 3′,5′‐diphosphate), it reacts very slowly with the 3′‐phosphate group. The enzyme does not hydrolyse adenosine 3′‐phosphate, 5′‐nucleotides and others phosphorylated substrates. The 5′‐phosphate group of the nucleotidic ribose and the type of puric base seem to play an important rôle in the formation of the enzyme‐substrate complex. The optimum pH of the 2′‐nucleotidase is around 5.5 and its Km value for NADP+ is between 0.7 to 1.0mM at pH 5.5 and between 1.4 to 1.8 at pH 7.0. The ions Na+, K+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+ have no influence on the enzymatic reaction; inorganic pyrophosphate, orthophosphate, adenosine, ADP and ATP are competitive inhibitors; NaF, Zn2+, Mo4+ and chiefly Cu2+ ions are strong non‐competitive inhibitors. The Ki of these effectors has been measured. It appears that these molecules, taking into account the concentration and the Ki‐value of ATP and Cu2+ in the latex, exert a physiologic effect on the 2′‐nucleotidase reaction. This enzyme of Hevea brasiliensis latex is probably an important factor in the mechanism of regulation of polysioprene synthesis.
Isolation and Characterisation of Two Calf‐Thymus Chromatin Non‐Histone Proteins with High Contents of Acidic and Basic Amino Acids
Authors: Goodwin Graham H. , Johns Ernest W.
Published: March 2005

The group of calf thymus chromatin non‐histone proteins which have a high electrophoretic mobility have been fractionated by CM‐cellulose ion‐exchange chromatography. The fractions thus obtained have been characterised by polyacrylamide gel electrophoresis, amino acid analysis, N‐terminal amino acid analysis, and by their molecular weights. Using the CM‐cellulose chromatographic method, two proteins which have a high content of basic and acid amino acids have been isolated in a pure form.
Fluorescence Study of Liver‐Alcohol‐Dehydrogenase Complexes with Berberine and Other Ligands
Authors: Kovář Jan , Skurský Ladislav
Published: March 2005

The dissociation constant of the enzyme · berberine complex has been determined by various fluorescence titration methods. The good agreement of the dissociation constants supplied by the titration of the complex by NADH, o‐phenanthroline, pelargonic and capric acids gives additional support for the previously made assumption that berberine binds to the active centre of the enzyme.

The lipophilic character of both partners is essential for the ability to bind. Nevertheless other results support the view that more specific interactions, possibly with the Zn atom and the –SH groups, are involved also.

A preliminary judgement about steric factors of the studied interaction is supplied by the ability of adenosine diphosphate‐ribose to form a ternary complex with the enzyme and berberine. Also NAD itself, although competing with berberine in lower concentrations, seems to form a ternary complex at high NAD concentrations. Both pyrazole and ethnol compete with berberine. The corresponding fluorescence titrations of the enzyme · berberine complex enable us to evaluate the dissociation constants of the binary complexes of the enzyme with pyrazole and alcohol.

Attempts have been done to measure the denaturation rates of the enzyme in acid media by following the disappearance of the fluorescent enzyme · berberine complex.
Lipid Requirement of Membrane‐Bound ATPase
Authors: Roelofsen Ben , van Deenen Laurens L. M.
Published: March 2005

The (Na++ K+)‐stimulated ATPase of human erythrocyte ghosts is completely inactivated by treatment with pure phospholipase A2 or phospholipase C.

These phospholipases also cause considerable or even complete loss of Mg2+‐dependent ATPase activity.

Enzymatic hydrolysis of sphingomyelin does not significantly influence either of the ATPase activities.

Conversion of the glycero‐phospholipids into phosphatidic acid by phospholipase D causes a drastic decrease in the Mg2+‐dependent ATPase activity, whereas the (Na++ K+)‐stimulated activity is increased.

Enzymatic decarboxylation of the phosphatidylserine fraction of the ghosts does not influence the (Na++ K+)‐stimulated ATPase, unless the last 13% of this fraction is converted into phosphatidyethanolamine, when there is a complete loss of this particular ATPase activity. On the other hand, the Mg2+‐dependent activity tends to be increased by this treatment.

The (Na++ K+)‐stimulated ATPase activity of erythrocyte ghosts treated with phospholipase C and subsequently extracted with dry ether, can be completely reconstituted by the addition of phosphatidylserine. Phosphatidylcholine, phosphatidylethanolamine and phospholipid mixtures are found to be ineffective. Phosphatidic acid produced a partial restoration of activity.

It is concluded that the (Na++ K+)‐stimulated ATPase activity strongly depends upon the presence of phosphatidylserine, of which only a minor fraction in the erythrocyte membrane is directly involved. It cannot be concluded from the experiments described in this paper that the Mg2+‐dependent ATPase also requires a specific phospholipid. However, a direct involvement of either cholesterol or sphingomyelin in this activity can be precluded.
The Anatomy of the Mitochondrial DNA
Authors: Porcher Harald H. , Koch Jürgen
Published: March 2005

The distribution of the mitochondrial DNA among the allomorphic forms, supercoiled circle, open circle and linear rod is a characteristic feature of each cell line. After pulse‐chase labeling with [Me‐3H]thymidine the bulk of the radioactivity can be located either in the supercoiled circular mitochondrial DNA (monkey Vero, ATCC; CCL 81) or in the open circular mitochondrial DNA (mouse NCTC 929, ATCC; CCL 1).

The open circular mitochondrial DNA from Vero cells contains one single nick, which is located in the heavy strand (alkaline CsCl gradient). In contrast, the open circular mitochondrial DNA from mouse cells contains one nick in one strand and at least two nicks in the opposite strand.

The bulk of the supercoiled Vero mitochondrial DNA has a heat‐labile site in the heavy strand and one site in both strands at which the phosphodiester backbone can be hydrolyzed by pancreatic RNAase.
Immunochemical Studies on Shigella dysenteriae Lipopolysaccharides
Authors: Dmitriev Boris A. , Backinowsky Leon V. , Lvov Vjacheslav L. , Kochetkov Nikolay K. , Hofman Irina L.
Published: March 2005

An investigation of the sugar composition of lipopolysaccharides derived from ten well‐defined Shigella dysenteriae serotypes and two Escherichia alcalescens‐dispar serotypes was carried out. All the lipopolysaccharides contained glucosamine, glucose, galactose, heptose and 3‐deoxyoctulosonic acid. These sugars were considered to represent the core polysaccharide and the same sugars except heptose and 3‐deoxyoctulosonic acid, may represent the constituents of the O‐specific polysaccharide chains. In addition to the “basal” sugars, the following monosaccharides were detected as components of the various Sh. dysenteriae lipopolysaccharides: galactosamine, mannose, fucose, rhamnose and 3,6‐dideoxyhexose. As a result, a chemical classification of Sh. dysenteriae lipopolysaccharides into eight chemotypes was suggested. The sugar composition of lipopolysaccarides from serologically related Shigella and Escherichia species was compared and several cross‐reacting pairs were found to belong to the same chemotype.
Irreversible Neonatal Differentiation of Corticosterone Metabolism in Rats in vivo
Authors: Begue René‐Jean , Gustafsson Jan‐Åke , Gustafsson Sven A.
Published: March 2005

The excretion of corticosterone metabolites in bile from gonadectomized male and female rats was studied; the effects of testosterone propionate treatment on steroid excretion in intact and castrated female rats were also studied. Postpubertal ovariectomy did not change the female biliary corticosteroid pattern (mainly characterized by excretion of mono‐ and disulphurylated 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one); likewise postpubertal testectomy or even testectomy of rats 14 days of age did not change the male biliary corticosteroid pattern (mainly characterized by disulphurylated 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 5α‐pregnane‐3β,11β,20β,21‐tetrol). On the other hand neonatal testectomy led to a feminized biliary corticosteroid pattern in the adult rats. The results indicate an irreversible suppression of the hepatic 15α‐hydroxylase activity by testicular secretion products, presumably androgen(s), in the neonatal period. When adult intact or gonadectomized female rats were treated with testosterone propionate the female biliary corticosteroid pattern changed to a male pattern but reverted to a female pattern some time after the therapy was stopped. These results indicate that the 15α‐hydroxylase activity in female rats may be reversibly suppressed by androgen treatment postpubertally.
On the Initiation of Transcription by DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: Schäfer Rolf , Krämer Reinhard , Zillig Wolfram , Cudny Henryk
Published: March 2005

It is shown that preinitiator or storage stretches on T5‐DNA are not a consequence of the existence of natural nicks in this template. Nicks do not contribute to the formation of heparinlabile and stable starters.

Analysis of the number of pppA and pppG starters and their products on T7‐DNA in enzyme saturation in the presence of heparin yielded 8 stable pppA starters, all synthesizing one product from the r‐strand sedimenting with 32 S and with a molecular weight of 1.9 × 106 (from sedimentation rate) or 2.2 × 106 (from chain length). Six labile pppG starters, yielding products from the l‐strand and, in addition, two very labile pppG starters seen only in the absence of heparin, also on the l‐strand, were demonstrated. These results give further evidence for the existence of preinitiator (or storage) stretches, with another template for which no assumption about the number of reading units have to be made.

The polyanion heparin is well suited for the elimination of artificial starts in vitro.
Glutamate déshydrogénase
Authors: Pantaloni Dominique , Lécuyer Bernard
Published: March 2005

The complexes which mammalian‐liver glutamate dehydrogenase forms with its coenzymes, substrates and effectors have been characterised by difference spectroscopy, optical‐rotatory‐dispersion and circular‐dichroism studies. Particular attention was paid to the enzyme · NADH · glutamate complex which was studied in the absence and in the presence of the effectors ADP and GTP.

Formation of the complexes is generally related both to modifications in the conformation of the enzyme and to the interaction between the enzyme and the coenzyme. Binding of the substrates to the NADH · enzyme complex induces changes in the circular‐dichroism spectrum of strates to the NADH · enzyme complex induces changes in the circular‐dichroism spectrum of the bound coenzyme: 2‐oxoglutarate increases the amplitude of the signal and glutamate an inversion of the spectrum.

The amount of the non‐productive complex enzyme · NADH · glutamate formed during the catalysis is very high as long as the concentration of 2‐oxoglutarate remains low in comparison with the concentration of glutamate.

There is one specific binding site for each ligand on each protomer (Mr= 56000) of the enzyme. Binding of the effectors induces no variation in the number of sites either of the coenzymes or of the substrates.

Besides binding to its own active site, NADH may also bind to the adenylic site of ADP. However, formation of the ternary complex enzyme · NADH · glutamate induces an increase in the affinity of NADH for its active site and an important decrease in its affinity for the adenylic site.

Competition for the catalytic site between NAD+ and NADH and between L‐glutamate and 2‐oxoglutarate has been confirmed by rotatory power studies.

It has been shown that GTP increases and ADP decreases the stability of the enzyme · coenzyme · substrate ternary complexes. GTP promotes the formation of the complex E · NADH · 2‐oxoglutarate while ADP promotes that of E · NADH · glutamate. These opposite effects of ADP and GTP are discussed in relation to the regulation of the enzymatic activity of glutamate dehydrogenase.
Studies on the Location and Orientation of Proteins in the Sarcoplasmic Reticulum
Authors: Thorley‐Lawson David A. , Green N. Michael
Published: March 2005

The adenosine triphosphatase of sarcoplasmic reticulum has been split into three parts by digestion with trypsin. A rapid split into fragments of molecular weight 60000 and 55000 was followed by a slow splitting of the 60000‐mol. wt piece into fragments of molecular weight 33000 and 24000. further digestion caused breakdown to small peptides and loss of protein from the vesicles.

Vesicles reconstituted from purified ATPase lipoprotein have the same appearance in the electron microscope and suffer the same specific digestions as do the sarcoplasmic reticular vesicles; we conclude, therefore, that the characteristic projections seen on vesicles of the sarcoplasmic reticulum are part of the ATPase molecule.

The two smallest fragments, molecular weight 33000 and 24000 were extensively iodinated by lactoperoxidase showing that they were exposed on the outer surface. Further, the phosphorylated active site has been located on the 33000‐mol. wt fragment. We suggest, therefore, that the 60000‐mol. wt fragment is responsible for the observed projections and that the active site is located in this projection and is outside of the membrane.

We have shown that calsequestrin is readily extracted from sarcoplasmic reticulum by EDTA and is significantly iodinated by lactoperoxidase. It is likely therefore, that this protein is located on the outer surface of the membrane.

Comparison of polyacrylamide gels of intact and digested sarcoplasmic reticulum suggests that several minor proteins of the sarcoplasmic reticulum may be proteolytic breakdown products of the ATPase.
Respiratory Control and the Proton Electrochemical Gradient in Mitochondria
Authors: Padan Etana , Rottenberg Hagai
Published: March 2005

The relationship between the rate of electron transport and the proton electrochemical gradient ΔH across the inner membranes of rat liver mitochondria was investigated. ΔH was calculated from ΔPH as measured by 14C‐labeled 5,5‐dimethyl‐2, 4‐oxazolidine dione distribution and from Δφ as measured by 42K distribution (in the presence of valinomycin). When mitochondria are in state 3, i.e. during phosphorylation, ΔH was only 5 m V less than in state 4, while respiration increased 3.5‐fold. In the presence of uncoupler, respiration was the same as in state 3, but ΔH was reduced by 22 m V with dinitrophenol, 28 m V with KCI + valinomycin and 60 m V with gramicidin. In the presence of valinomycin, when uncoupling increases by increasing KCl concentration, there was a linear relationship between ΔH and the rate of respiration. These results are in agreement with the suggestion that in uncoupled systems the respiratory rate is controlled by ΔH. However, the phosphorylation reaction controls the respiratory rate directly and not through its effect on ΔH. This conclusion is not compatible with the chemiosmotic model of oxidative phosphorylation.
Determination of the Dissociation Constants of the Lysine Residues of Lysozyme by Proton‐Magnetic‐Resonance Spectroscopy
Authors: Bradbury J. Howard , Brown Larry R.
Published: March 2005

Amino groups of a series of model compounds and proteins are methylated by reductive condensation with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance experiments. The extent of methylation varies from 92% for ribonuclease A to 66% for bovine serum albumin. As observed by PMR difference spectroscopy, the conformation of most of these proteins is unaltered by the modification. Retention of native conformation by methylated lysozyme is also shown by enzymatic activity measurements and PMR observation of thermal denaturation.

Resonances corresponding to each of the six Nɛ‐dimethyllysine residues in methylated lysozyme are resolved at 270 MHz and titrated to obtain individual pK values. A correction factor of 0.6 pH units obtained from model compound studies is applied to obtain apparent pK values ranging from 10.1 to 10.7 for the ɛ‐amino groups of lysozyme. Assignment of resonances to specific residues is accomplished using the selective broadening effect of Gd3‐. These results indicate the utility of the N‐dimethyl group for probing the environment of lysine residues in proteins.
Phylogeny of the Neurohypophysial Hormones
Authors: ACHER Roger , Chauvet Jacqueline , Chauvet Marie‐Thèrèse
Published: March 2005

The neurohypophysial hormones of a chondrostean, the sturgeon (Acipenser sp.), have been purified by adsorption onto neurophysin, dissociation of the complex hormone‐protein by trichloroacetic acid precipitation and isolation of active peptides, from the supernatant solution, by paper chromatoelectrophoresis.

Arginine vasotocin has been characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties as well. The amount of arginine vasotocin (about 50 nmol per g pituitary powder) is intermediary between those found for bony fishes (about 1 000 nmol/g) and cartilaginous fishes (about 5 nmol/g).

A second hormone, which can be classified in the oxytocin‐like type by its electrophoretic migration and its pharmacological properties, has been disclosed. The very weak amount did not allow chemical identification. However the chromatographic behaviour and the pharmacological “profile” indicate that this hormone differs from the six known oxytocin‐like peptides.
Subunit Structure of Aspartokinase III of Escherichia coli K12
Authors: Richaud Catherine , Patte Jean‐Claude , Mazat Jean‐Pierre , Gros Claude
Published: March 2005

The lysine‐sensitive aspartokinase III of Escherichia coli K12 is composed of subunits of 50000 molecular weight. By determination of terminal residues and inspection of tryptic hydrolysis products, the subunits appear identical. The native enzyme is a dimer of 105000 molecular weight in the presence of 0.15 M KCl in the absence of lysine. Two types of equilibrium can be evidenced: an association at high ionic strength in the presence of lysine leading to a tetrameric form and a dissociation towards monomer at low ionic strength in the absence of lysine. The two equilibria are dependent on protein concentration.
Basic Structure of Mouse Histocompatibility Antigens
Authors: Hess Maxime , Davies D. Allen L.
Published: March 2005

Mouse histocompatibility antigens were solubilized from lymphocyte membranes by limited papain degradation. Spleens from Balb/c mice (H‐2d), enlarged by administration of lymphoma cells, were used for the membrane preparations. The solubilized protein components were purified by ion‐exchange chromatography, gel filtration and discontinuous polyacrylamide‐gel electrophoresis. A further characterization was achieved in a two‐dimensional protein mapping system using disc‐electrophoresis and isotachophoresis in the first and second dimension, respectively. The purification was monitored for alloantigens H‐2.4(D) and H‐3.21(K) (H‐2D and H‐2K represent two regions of the H‐2 system). On Sephadex G‐100 partially purified H‐2‐alloantigen chromatographed in a region of Mr= 35500. The pI of these substances showed a maximum at pH 5.5 and 5.3 for H‐2.4 and at 5.2 for H‐2.31. Free SH‐ and dithio‐groups were determined with 5,5′‐dithiobis(2‐nitrobenzoic acid). 1.56 μmol SH/μmol alloantigenic material was found, but no SS‐bond could be detected after alkaline cleavage. This suggests the absence of covalently linked protein subunits in papain‐solubilized molecules bearing single private specificites. This was confirmed after reduction and alkylation in 4 M urea: 30–50% of the alloantigenic activity was retained and shown, in polyacrylamide‐gel electrophoresis, to migrate in the original region of activity (RBPB 0.29 and 0.33 for H‐2.4; about 0.33 for H‐2.31, BPB = bromophenol blue). After reductive alkylation gel filtration resulted in a further purification of the alloantigenic material. Highly purified substances showed a diffuse staining pattern and a broad serological profile after electrophoresis. We assume this heterogenity to be a genuine property of H‐2 antigens. Structural and genetic implications of these findings are discussed.
Studies on the Catalytic Mechanism of a Serine Protease from Streptomyces griseus
Authors: Bauer Carl‐Axel , Löfqvist Bo , Pettersson Gösta
Published: March 2005

The kinetics of the Streptomyces griseus protease‐3‐catalyzed hydrolysis of p‐nitrophenyl‐acetate have been studied by stopped‐flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three‐step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl‐enzyme and a rate‐limiting deacylation of this intermediate. Estimated rate constants forthe latter two reaction steps are 15 s−1 and 0.09 s−1, respectively.

The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady‐state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate‐limiting deacylation step in the catalytic mechanism.