bluuebunny/crossref_metadata_2025 huggingface dataset:
Hysteretic Behaviour of Citrate Synthase. Alternating Sites during the Catalytic Cycle
Authors: LÖHLEIN‐WERHAHN Gerlinde , BAYER Ernst , BAUER Barbara , EGGERER Hermann
Published: March 2005

Chemically and stereochemically pure (3S)‐citryl‐CoA was prepared enzymically and used as a substrate for citrate synthase to investigate the previously determined unexpectedly low rate of hydrolysis of the (3 RS)‐substrate. The unnatural R‐diastereomer of this mixture is not inhibitory.

At low enzyme concentrations the rate of citryl‐CoA hydrolysis was linear until the reaction went near to completion; the hydrolysis approached Michaelis‐Menten kinetics at high enzyme concentrations. In between these concentration extremes a biphasic rate dependence was detectable, where a fast initial phase lasting a few seconds was followed by a slow steady‐state phase.

Citrate synthase was characterized as a hysteretic enzyme existing in two interconvertible forms, which were designated according to their functions as hydrolase E and ligase E'.

The hysteretic behaviour originates in the cleavage of citryl‐CoA to acetyl‐CoA and oxaloacetate. This reaction occurs on the ligase form E', which represents a trap for enzyme form E, the hydrolase.

The conclusions given above are strengthened by the ordinary hydrolysis kinetics of (2S)‐malyl‐CoA, a substrate that is not subject to cleavage of the C‐C bond on the synthase.

The results satisfy the kinetic criterion for citryl‐CoA being an intermediate of the physiological synthase reaction and, therefore, establish the oscillation of the synthase between hydrolase and ligase states during the catalytic cycle.

A disorganization of these oscillations can be achieved by limited tryptic proteolysis of the synthase.
Porphyran Primary Structure
Authors: MORRICE Lora M. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B.
Published: March 2005

Porphyran, a highly substituted agarose from Porphyra umbilicalis was degraded by highly purified β‐agarase I from Pseudomonas atlantica. This enzyme cleaved at the reducing side of units of β‐neoagarobiose (3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐β‐d‐galactopyranose).

The oligosaccharides were divided into fractions of low and high molecular weight by dialysis. The permeate (23% of total starting carbohydrate) was separated by ion‐exchange into neutral and anionic fractions. Gel filtration of the neutral fraction (19%) resolved two major oligosaccharides. These were shown by 13C‐NMR spectroscopy to be 63‐O‐methyl‐neoagarotetraose and 63,65‐di‐O‐methyl‐neoagarohexaose. Gel filtration of the anionic oligosaccharides (3.3%) revealed two novel monosulphated tetrasaccharides, 6‐O‐sulphato‐α‐l‐galactopyranosyl‐(1 → 3)‐β‐d‐galactopyranosyl‐(1 → 4)‐3,6‐anhydro‐α‐l‐galactopyranosyl‐(1 → 3)‐d‐galactopyranose and its 63‐O‐methylated derivative.

The 13C‐NMR data from the sulphated tetrasaccharides provided a novel reference which was used to characterise higher, partially sulphated fragments in the dialysis permeate. The fraction retained on dialysis (77%) had an average degree of polymerisation of 40 and was homologous with the high‐molecular‐weight anionic permeate. From 13C‐NMR spectroscopy porphyran was found to comprise 49% sulphated disaccharide units and these were calculated to occur in stretches averaging 2.0–2.5 contiguous units.
Brain Proteolipids
Authors: HELYNCK Gérard , LUU Bang , NUSSBAUM Jean‐Louis , PICKEN Douglas , SKALIDIS Georges , TRIFILIEFF Elisabeth , Van DORSSELAER Alain , SETA Patrick , SANDEAUX Roger , GAVACH Claude , HEITZ Frédéric , SIMON Daniel , SPACH Gérard
Published: March 2005

Proteolipid apoproteins have been isolated from a whole bovine brain homogenate by chloroform/methanol extraction, and fractionated by chromatography on modified (lipophilic) Sephadex, followed by ion‐exchange chromatography on CM‐Trisacryl. The various final, highly hydrophobic, fractions are homogeneous (sodium dodecyl sulfate/polyacrylamide gel electrophoresis). Transmembrane ion transfers were studied by22Na+flux and electrical conductance measurements. Single channel events were observed at low protein concentrations, in particular with one of the final homogeneous apoproteolipids of molecular mass 24 kDa.
The Use of Alginate Ester Films on Solid Supports in the Preparation of Water‐Insoluble Immunoadsorbents for Purification of Antigens
Authors: KENNEDY John F. , BARNES Junor A. , BARKER S. Alan
Published: March 2005

A novel method developed for the preparation of immunoadsorbents is based on the technique of coating potential column supports with a propylene glycol alginate ester film created by emulsion polymerisation. The cross‐linking of the film with 1,2‐diaminoethane in alkaline solution resulted in a water‐permeable but water‐insoluble film which was stable at high pH. Sheep anti‐(human IgG) was subsequently coupled to the cyanogen‐bromide‐activated film and the resultant immunoadsorbents were used to purify IgG from whole human serum. The production of the film‐coated supports, chemical coupling of antibody and the chromatographic characteristics of four alginate‐film‐coated supports are described. With the use of Sepharose®– alginate‐ester–sheep‐anti‐(human IgG) immunoadsorbent the overall yield for IgG from five serial adsorption‐elution cycles was 94% of the adsorbed IgG. This immunoadsorbent proved to be of high binding capacity and allowed the elution of specific antigen in almost 100% yield and high purity with minimal non‐specific interference from other serum proteins.
Purification and Partial Characterization of a Highly Immunogenic Human Leukocyte Protein, the L1 Antigen
Authors: DALE Inge , FAGERHOL Magne K. , NAESGAARD Inger
Published: March 2005

L1 is a major and highly immunogenic protein component of human granulocytes. It was purified from random samples of blood‐donor leukocytes. An antiserum to L1 was initially raised in rabbits by immunization with protein fractions from preparative agarose gel electrophoresis. The main problem in purifying L1 was its poor stability when carried through multiple steps. Preparative isoelectric focussing was therefore adopted as an efficient one‐step method. The isoelectric focussing pattern of L1 was greatly influenced by the presence of EDTA or calcium ions in the sample buffer. With low EDTA concentrations or calcium excess, L1 focussed as seven protein bands in two regions, pH 6.1–6.5 and pH 7.6–8.4. Conversely, L1 was found only in the pH‐6.1–6.5 region when excess EDTA was added to the sample. Irrespective of conditions, the bulk of L1 focussed at pH 6.3 and pH 6.5. Also the electrophoretic mobility of L1 was strongly influenced by calcium ions, suggesting uptake of calcium by the protein. In the presence of calcium, L1 adhered to glass surfaces and filters used for concentration of protein solutions. The latter problem could be prevented by addition of EDTA. The molecular mass of L1 was determined to be about 36.5 kDa. The molecule was shown to consist of three non‐covalently linked 12.5‐kDa subunits.
Characterization of Cytochrome P2‐450 (20‐S) mRNA
Authors: IKEDA Toshihiko , ALTIERI Mario , CHEN Yuan‐Tsong , NAKAMURA Michitoshi , TUKEY Robert H. , NEBERT Daniel W. , NEGISHI Masahiko
Published: March 2005

Mouse liver cytochrome P2‐450 is defined as the major isosafrole‐inducible form of P‐450 which is most specific for isosafrole metabolism. λAhP‐1 represents a 15.5 × 103‐base‐pair segment of mouse genomic DNA having the cytochrome P1‐450 gene (∼ 4600 base pairs) located in the middle portion. Using various subclones as probes, we investigated the differential expression of P1‐450 mRNA and P2‐450 mRNA induction as a function of association with the Ah locus, 3‐methylcholanthrene or isosafrole dosage, tissue specificity, and developmental age.

Both P1‐450 (23‐S) mRNA and P2‐450 (20‐S) mRNA induction processes are regulated by the Ah receptor. P2‐450 mRNA is about 10‐fold more sensitive than P1‐450 mRNA to induction by either 3‐methylcholanthrene or isosafrole. Phenobarbital pretreatment has no effect at all on either P1‐450 mRNA or P2‐450 mRNA. Whereas both P1‐450 mRNA and P2‐450 mRNA are induced by 3‐methycholanthrene in C57BL/6N liver, P1‐450 (23‐S) mRNA but not P2‐450 (20‐S) mRNA is induced by 3‐methylcholanthrene in C57BL/6N kidney. P1‐450 mRNA induction by 3‐methylcholanthrene is measurable in C57BL/6N liver at day 15 of gestation, and the expression becomes enhanced with increasing age. P2‐450 mRNA induction by 3‐methylcolanthrene in C57BL/6N liver appears about 7 days later during development than 3‐methylcholanthrene‐inducible P1‐450 mRNA. Both 3‐methylcholanthrene‐induced P1‐450 mRNA and P2‐450 mRNA are detectable in DBA/2N liver; their appearance is later in development, however, and at lower concentrations than that seen with C57BL/6N liver. P1‐450 (23‐S) mRNA and P2‐450 (20‐S) mRNA appear to hybridize to a common 5′ fragment of the P1‐450 gene.
Comparative Receptor Study in Gamete Chemotaxis of the Seaweeds Ectocarpus siliculosus and Cutleria multifida
Authors: BOLAND Wilhelm , MARNER Franz‐Josef , JAENICKE Lothar , MÜLLER Dieter G. , FÖLSTER Elsbet
Published: March 2005

Gamete fusion in phaeophytes is initiated by olefinic hydrocarbons as messenger substances. They are secreted by the female gametes and act on male gametes or gametangia. The sensitivity thresholds of the androgametes of Ectocarpus siliculosus and Cutleria multifida, two brown algae of different evolutionary stages, for ectocarpene, which is produced by either species, were found to be 0.89 nmol/l and 8.9 nmol/l sea water, respectively. The molecular recognition mechanism is shown to be identical for these two species and probably also for most other phaeophytes. The pheromones involved are structurally related and sometimes resemble each other in their overall π‐electron distribution (e.g. ectocarpene and multifidence). Knowing the specific messenger, the typical response behaviour of each receptor involved, the pheromone productivity of the considered species as well as other biological parameters (phytogeographic distribution, the natural benthic habitat and times of fertilization), a method is presented to uncover possible interspecific cross‐reactions of such algal communication system as a ‘chemical warfare’ device in their fight for living space.
Exchange of Histones H1 and H5 between Chromatin Fragments
Authors: THOMAS Jean O. , REES Christine
Published: March 2005

Histones H1 and H5 can exchange between an H1‐containing chromatin fragment from rat liver and an H1, H5‐containing fragment from chicken erythrocytes at ionic strengths from about 35 mM to 105 mM. The redistribution has reached equilibrium by ionic strength 75 mM in 1 h or less at 4°C.

After exchange at ionic strength 75 mM, long fragments, whether of rat liver or chicken erythrocyte chromatin, are recovered with a higher H5:H1 ratio than short fragments, suggesting a stronger preference of H5 than of H1 for higher‐order structures which exist for long fragments at ionic strength 75 mM.

Competition experiments between occupied H1 or H5 binding sites on chromatin fragments from rat liver or chicken erythrocytes and empty sites on H1‐depleted rat chromatin show that rat H1 does not distinguish between the two types of site, whereas H5 discriminates in favour of sites on native chromatin, even when the chicken fragments are too short to form higher‐order structures. (The behaviour of the chicken H1, which may be bound less tightly than rat H1, depends on the length of the chicken fragment, in a manner suggesting that fragments of 15 nucleosomes and longer can form stable higher‐order structures which have high‐affinity binding sites for both H5 and H1.)

We conclude that the affinity of sites for H5 is in the order: higher‐order structures > nucleosome filament ≫ H1‐depleted chromatin. The same relative order of affinities may well apply for H1 but the discrimination is much lower. This difference between H1 and H5 seems likely to be relevant to the greater stability of H5‐containing chromatin, and in turn its transcriptional inactivity, and indeed to the mechanism of replacement of H1 by H5 during the terminal stages of erythropoiesis.
Identification of new Sialic Acids Derived from Glycoprotein of Bovine Submandibular Gland
Authors: REUTER Gerd , PFEIL Reinhard , STOLL Sabine , SCHAUER Roland , KAMERLING Johannis P. , VERSLUIS Cornelis , VLIEGENTHART Johannes F. G.
Published: March 2005

Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N‐acetyl‐8‐O‐acetylneuraminic acid, N‐acetyl‐8,9‐di‐O‐acetylneuraminic acid, N‐acetyl‐7,8,9‐tri‐O‐acetylneuraminic acid, N‐glycoloyl‐7‐O‐acetylneuraminic acid, N‐glycoloyl‐7,9‐di‐O‐acetylneuraminic acid, N‐glycoloyl‐8,9‐di‐O‐acetylneuraminic acid, and N‐glycoloyl‐7,8,9‐tri‐O‐acetylneuraminic acid. There are also indications for the presence of N‐glycoloyl‐8‐O‐acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N‐acetylneuraminic acid, N‐acetyl‐7‐O‐acetylneuraminic acid, N‐acetyl‐9‐O‐acetylneuraminic acid, N‐acetyl‐7,9‐di‐O‐acetylneuraminic acid, N‐glycoloyl‐neuraminic acid, and N‐glycoloyl‐9‐O‐acetylneuraminic acid could be identified.

Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anionexchange column (formate form) by elution with a 0–0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n‐butanol/n‐propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N‐acetylneuraminic acid, N‐acetyl‐7‐O‐acetylneuraminic acid, N‐acetyl‐9‐O‐acetylneuraminic acid, N‐glycoloylneuraminic acid, and N‐glycoloyl‐9‐O‐acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one‐dimensional and two‐dimensional thin‐layer chromatography, gas‐liquid chromatography, gas‐liquid chromatography, and gas‐liquid chromatography–mass spectrometry.
Reduction of Cytochrome P‐450 LM2 by NADPH in Reconstituted Phospholipid Vesicles is Dependent on Membrane Charge
Authors: INGELMAN‐SUNDBERG Magnus , BLANCK Jürgen , SMETTAN Gerhard , RUCKPAUL Klaus
Published: March 2005

The kinetics of the reduction of cytochrome P‐450 LM2 mediated by NADPH‐cytochrome P‐450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of the reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P‐450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O‐demethylation of p‐nitroanisole catalyzed by P‐450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH‐cytochrome P‐450 reductase and cytochrome P‐450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.
Different Forms of α2u‐Globulin in Male and Female Rat Urine
Authors: VANDOREN Gaby , MERTENS Bea , HEYNS Walter , VAN BAELEN Hugo , ROMBAUTS Wilfried , VERHOEVEN Guido
Published: March 2005

α2u‐Globulin is usually considered to be present only in male rat urine. This study demonstrates that a very similar protein exists in female rat urine and compares its properties with those of the male form. Isoelectric focusing followed by immunofixation reveals considerable microheterogeneity of α2u‐globulin in male and female rat urine. Important sex differences are noted in the banding pattern. The isoelectric point of the major male component (pI ∼ 5.3) is considerably higher than that of the major female components (pI ∼ 4.6). In addition, the female form of α2u‐globulin has a somewhat higher mobility on sodium dodecyl sulphate/polyacrylamide gel electrophoresis than its male counterpart. These sex differences are preserved after purification of α2u‐globulin from male and female rat urine by affinity chromatography and enrichment of the major male and female components by ion‐exchange chromatography. Immunologically no differences are observed between these purified components and their amino acid composition reveals only minor differences. A slightly higher carbohydrate content is observed in the major female component than in the major male component. Finally evidence is presented that oestrogen treatment suppresses the male forms of α2u‐globulin but has no effect on the female forms. The observed differences between the male and female forms and their different hormonal control suggest that they are encoded by different genes.
The Metabolism of Chenodeoxycholic Acid to β‐Muricholic Acid in Rat Liver
Authors: BOTHAM Kathleen M. , BOYD George S.
Published: March 2005

The synthesis of conjugated chenodeoxycholic acid and tauro‐β‐muricholic acid in isolated rat hepatocytes was measured by radioimmunoassay. Production of tauro‐β‐muricholic acid was linear over 4 h of incubation at 37°C. The net synthesis of conjugated chenodeoxycholic acid was very much lower than that of tauro‐β‐muricholic acid.

When hepatocytes were prepared from rats in which the enterohepatic circulation had been broken, either by feeding the bile salt sequestrant, cholestyramine or by total biliary drainage for 48 h, synthesis of tauro‐β‐muricholic acid was increased compared to that in cells from control rats. Conjugated chenodeoxycholic acid accumulation during incubation of the hepatocytes was increased by cholestyramine feeding but not by total biliary drainage. These results suggest that there is a metabolic difference between the two methods of breaking the enterohepatic circulation with regard to chenodeoxycholic acid synthesis.

Hepatocytes prepared from rats given 1% cholesterol in the diet for at least 2 weeks synthesised significantly more tauro‐β‐muricholic acid than those from control rats. The total amount of conjugated cholic, chenodeoxycholic and tauro‐β‐muricholic acids synthesised by cells from cholesterol fed animals, however was not significantly different from that produced by hepatocytes from normal rats.

Exogenous taurochenodeoxycholic acid was metabolised to tauromuricholic acid by isolated hepatocytes. Production of tauro‐β‐muricholic acid reached a maximum at a concentration of 20 μM taurochenodeoxycholic acid. The total metabolism of taurochenodeoxycholic acid, however, increased linearly up to the highest concentration measured, 50 μM.

The biliary content of tauro‐β‐muricholic acid during total biliary drainage fell rapidly in the first 10 h and thereafter continued to decline, reaching a minimum after about 24 h. No significant rise was observed during the remainder of the experimental period.

It is concluded that a large proportion of the conjugated chenodeoxycholic acid synthesised by isolated hepatocytes in vitro is metabolised to tauro‐β‐muricholic acid, and therefore it is necessary to take this into account when using this system to study bile salt synthesis.
Mechanism of the Increase in Cytochrome c Oxidase Activity in Pea Cotyledons during Seed Hydration
Authors: MATSUOKA Makoto , ASAHI Tadashi
Published: March 2005

The increase in cytochrome c oxidase activity in pea cotyledons during seed hydration was investigated with two kinds of antibodies against the enzymes purified from mitochondria of pea shoot and sweet potato root. The antibody against the pea enzyme recognized only the largest of the enzyme subunits (subunit I), while that against the sweet potato one did other three of the pea enzyme subunits: the second largest (subunit II) and the two smallest (subunits IV and V).

There was no increase in the amount of protein immunoprecipitated with the anti‐(pea enzyme) antibody in a crude fraction of cotyledon membranes during seed hydration, despite the marked increase in cytochrome c oxidase activity. Neither cycloheximide nor chloramphenicol had any effect on the activity or the amount. The crude membrane fraction from dry cotyledons contained an enzymatically inert protein precipitated with the anti‐(pea enzyme) antibody. The inert protein could be separated from the active enzyme protein through solubilization of the protein from the membranes with various concentrations of Triton X‐100. It consisted of only one kind of polypeptide with the same molecular weight as subunit I and decreased with the concomitant increase in the active enzyme protein during seed hydration.

Dry pea cotyledons also contained a soluble protein that was immunoprecipitated by antibody to the sweet potato enzyme. The soluble form of the immunoreactive protein decreased with the concomitant increase in active cytochrome c oxidase protein in the crude membrane fraction from the cotyledons during seed hydration. None of these changes during seed hydration were inhibited by cycloneximide or chloramphenicol. The soluble, immunoreactive protein contained only one kind of polypeptide, of which the molecular weight was identical to that of subunit IV.

The mitochondrial inner membranes of dry cotyledons were separated into three fractions by sucrose density gradient centrifugation. The lightest was the richest of the three in the enzymatically inert protein immunoprecipitated with the anti‐(pea enzyme)antibody; cytochrome c oxidase activity in this fraction increased immediately after the onset of seed hydration.

These results indicate that a membrane‐bound, free form of subunit I and a soluble, free form of subunit IV of cytochrome c oxidase exist in dry pea cotyledons. We propose that these free forms of subunits are assembled with the other subunits to form the active cytochrome c oxidase during seed hydration.
Identification of a Macromolecular Inhibitol of Glucocorticoid‐Receptor Complex Activation in Rat Liver Cytosol
Authors: BARNETT Carol A. , SPECK Lynn , LITWACK Gerald
Published: March 2005

We have identified and endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE‐cellulose. The macromolecular regulator, purified approximately 20‐fold as judged by Lowry‐reactive material, inhibits activation of glucocorticoid‐receptor complexes when assayed by DNA‐cellulose binding and by chromatography on DEAE‐cellulose minicolumns. In addition the active DEAE‐cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE‐cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90°C (15 min) and is partially inactivated at 45°C (15–60 min).
Purification and Properties of an Altered Form of Elongation Factor 2 from Mutant Cells Resistant to Intoxication by Diphtheria Toxin
Authors: IGLEWSKI Wallace J. , LEE Hebert
Published: March 2005

Elongation factor 2 (EF‐2) was isolated from wild‐type and mutant‐polyoma‐virus‐transformed baby hamster kidney cells resistant to intoxication by diphtheria toxin. Cells were grown as tumors in hamsters and EF‐2 was purified from tissue homogenates by column chromatography. Both forms of EF‐2 chromatograph identically in Whatman DE‐52 DEAE‐cellulose, Sephadex DEAE‐A50 and Sephacryl S‐200 resins. However, wild‐type and the mutant form of EF‐2 elute from phosphocellulose at 0.16 M and 0.24 M KCl respectively. Both forms of EF‐2 migrate in sodium dodecyl sulfate/polyacrylamide gels as a single band with an Mr of 93000 and produce identical 125I‐labeled tryptic peptide maps. However, additional labeled tryptic peptides are seen when wild‐type EF‐2 is ADP‐ribosylated by fragment A of diphteria toxin. The purified mutant protein is totally resistant to ADP‐ribosylation and cannot be transformed into an ADP‐ribosylatable form in a posttranslational modification system in vitro, indicating that resistance to ADP‐ribosylation results from a mutation in the structural gene for EF‐2.
Reaction of Monosubstituted Hydrazines and Diazenes with Rat‐Liver Cytochrome P450
Authors: BATTIONI Pierrette , MAHY Jean‐Pierre , DELAFORGE Marcel , MANSUY Daniel
Published: March 2005

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Soret peak at 446 nm. Upon oxidation of the [cytochrome P450‐Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex charaterized by a Soret peak at 486 nm is formed. The latter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450‐Fe(III) or in situ oxidation of methylhdrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO.

Reactions of ethyldiazene or benzyldiazene with cytochrome P450‐Fe(III) afforded similar complexes characterized by Soret peaks around 480 nm.

These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN = NH with hemoglobin and iron‐porphyrins, are consistent with a [cytochrome P450‐Fe(II)(RN = NH)] structure for the 446‐nm‐absorbing complexes and a σ‐alkyl cytochrome P450‐Fe(III)‐R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a σ‐cytochrome P450‐Fe(III)‐Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450‐Fe(III)‐R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines.
Primary Substrate Specificity Determinants for the H4‐Specific Protease‐Activated Protein Phosphotransferase
Authors: ECKOLS T. Kris , THOMPSON Richard E. , MASARACCHIA Ruthann A.
Published: March 2005

The specificity of the histone‐H4‐specific, protease‐activated protein kinase (H4‐PK) was examined using two series of synthetic peptides corresponding to the phosphorylation sites in histone H4 and pyruvate kinase. Optimum kinetic constants for phosphorylation were observed using the peptide Val‐Lys‐Arg‐Ile‐Ser‐Gly‐Leu. Peptides in which the Lys was replaced by Arg or the Lys‐Arg sequence was transposed were phosphorylated with less favorable kinetics. Peptides with either basic residue deleted did not serve as substrates. Only the H4 peptide, containing an Arg‐Arg sequence, was phosphorylated by the cyclic‐AMP‐dependent protein kinase (CA‐PK). Distinct specificity determinants for H4‐PK and CA‐PK were also observed using the pyruvate kinase peptide (Leu‐Arg‐Arg‐Ala‐Ser‐Leu‐Gly). Collectively the data indicated that the primary substrate specificity determinants for H4‐PK are Lys‐Arg‐Xaa‐Ser whereas the CA‐PK selectively phosphorylates the sequence Arg‐Arg‐Xaa‐Ser.
Isolation from Trout Liver of a Methionine‐Containing H1 Subfraction, THImet
Authors: BROWN Elizabeth , GOODWIN Graham H.
Published: March 2005

A trout H1 subfraction, THImet, which contains one residue of methionine and has a blocked N‐terminal amino acid, has been isolated from trout liver. Cyanogen bromide cleavage of THImet and amino acid analysis and polyacrylamide gel electrophoresis of the cleavage products show that it contains a residue of methionine at about the same distance from the N terminus as mammalian H1° and avian H5. THImet is shown to be similar to, but not identical with, the protein derived from trout erythrocytes generally accepted as the equivalent in fish of avian H5.
On the Mechanism of Inhibition of Neutral Liver Fructose 1,6‐Bisphosphatase by Fructose 2,6‐Bisphosphate
Authors: FRANÇOIS Jean , VAN SCHAFTINGEN Emile , HERS Henri‐Géry
Published: March 2005

The inhibitory effect of fructose 2,6‐biphosphate on fructose 1,6‐bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly non‐competitive inhibitor. The effect of fructose 2,6‐bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (α+β)fructofuranoside 1,6‐bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6‐bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (α+β)fructofuranoside 1,6‐bisphosphate. These observations can be explained by assuming that fructose 2,6‐bisphosphate has no affinity for the active site of fructose 1,6‐bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6‐bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.
Isotope‐Exchange Evidence that Glucose 6‐Phosphate Inhibits Rat‐Muscle Hexokinase II at an Allosteric Site
Authors: GREGORIOU Mary , TRAYER Ian P. , CORNISH‐BOWDEN Athel
Published: March 2005

The flux ratio for hexokinase type II from rat muscle, i.e. the rate of conversion of glucose 6‐phosphate molecules into ATP molecules divided by the simultaneous rate of conversion of glucose 6‐phosphate molecules into glucose molecules, increases with the MgATP concentration but is independent of the glucose concentration. This behaviour requires that glucose must bind before MgATP when the reaction is proceeding in the normal physiological direction, i.e. phosphorylation of glucose. Although at low non‐inhibitory glucose 6‐phosphate concentrations the flux ratio increases linearly with the MgATP concentration, the dependence becomes non‐linear, with a slope that increases with the MgATP concentration, at glucose 6‐phosphate concentrations above 1 mM. This behaviour does not permit glucose 6‐phosphate to act only as a normal product inhibitor. Instead, it seems to require glucose 6‐phosphate to act as an all osteric inhibitor and for a second site for binding of MgATP to exist. Measurements of the flux from ATP to glucose 6‐phosphate and to ADP showed no dependence of the flux ratio on the concentrations of either glucose 6‐phosphate or ADP. This result does not permit the order of product‐release steps in this direction to be determined, but shows that the second product is released virtually instantaneously after the first.
The Interactions between Monovalent Ions and Phosphatidyl Cholines in Aqueous Bilayers
Authors: SÖDERMAN Olle , ARVIDSON Gösta , LINDBLOM Göran , FONTELL Krister
Published: March 2005

The interactions of lithium and sodium ions and water with phosphatidylcholine bilayers have been studied by means of 7Li, 23Na and 2H NMR quadrupole splittings. The experimental results are interpreted in terms of a simple three‐site model with two anisotropic sites (‘binding’ sites) and one isotropic site (‘free’ ions and water molecules). The findings obtained for the zwitterionic model membrane are compared with previous investigations of lamellar phases composed of ionic and nonionic amphiphiles. It is shown that the data obtained are compatible with our previous suggestion [Lindblom, G., Persson, N.‐O., and Arvidson G. (1976) Adv. Chem. Ser. 152, 121] that an increase in the salt content in the water layer induces a conformational change in the polar head group of phosphatidylcholine. Thus at high salt concentration the phosphocholine head group tends to be oriented perpendicular to the lipid bilayer surface. The study also shows that increasing the amount of salt leads to a squeezing out of water between the bilayers. This is interpreted in terms of a reduction of the repulsion forces between the bilayers.
Saccharomyces cerevisiae cdc9, a Structural Gene for Yeast DNA Ligase which Complements Schizosaccharomyces pombe cdc 17
Authors: BARKER David G. , JOHNSTON Leland H.
Published: March 2005

The Saccharomyces cerevisiae cdc9 gene has been cloned in the vector YRp12 by complementation of the temperature‐sensitive lesion in vivo. The gene is contained within a 3300‐base‐pair fragment of DNA, which maps to the chromosomal locus of cdc9 and which is able to complement a DNA‐ligase‐deficient mutant of the fission yeast Schizosaccharomyces pombe.
Synthesis and Application of Two Reagents for the Introduction of Sulfhydryl Groups into Proteins
Authors: MAASSEN J. Antonie , THIELEN Ton P.G.M. , MÖLLER Wim
Published: March 2005

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate‐oxidized N termini of proteins, provided that the N‐terminal residue is serine or threonine. 3‐(Phenyldithio) propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.
Utilization of Energy Stored in the Form of Na+ and K+ Ion Gradients by Bacterial Cells
Authors: BROWN Igor I. , GALPERIN Michael Yu. , GLAGOLEV Alexey N. , SKULACHEV Vladimir P.
Published: March 2005

The hypothesis that Na+ and K+ gradients have an energy storing function [V. P. Skulachev (1978) FEBS Lett. 87, 171–176] has been tested in experiments with Escherichia coli, the marine bacterium Vibrio harveyi, an extremely halophilic Halobacterium halobium and a fresh‐water cyanobacterium Phormidium uncinatum from Lake Baikal living at an extermely low salt concentration. The capability of these microorganisms to maintain was compared using motility as a ‐supported function. It was found that in all cases the gradient of monovalent cations is competent to prolong the period of active motility after other energy sources are exhausted. Maximal prolongation was found in H. halobium, which in a Na+ medium was still motile when light was switched off for 9 h under anaerobic conditions. In V. harveyi the motility was maintained for 1 h, in E. coli for about 10 min and in Ph. uncinatum for about 2 min. Thus the buffer capacity of the monovalent cation gradient is proportional to the content of these cations in the habitat. It was also found that in Ph. uncinatum only ΔpK is effective, whereas in E. coli and V. harveyi both ΔpK and ΔpNa are.

In E. coli when the K+ release is completed and the cells become motionless, motility can be temporarily restored by adding NaCl which initiates an H+ efflux.

Under conditions of exhaustion of energy sources, the Na+ and K+ gradient was shown to stabilize potential in H. halobium cells, measured with a tetraphenylphosphonium probe.

In H. halobium and E. coli, the anaerobic ATP level was found to stabilize when the Na+ and K+ gradients were present. Addition of N, N′‐dicyclohexylcarbodiimide destabilized this level, which indicated that Na+ and K+ gradients could support de novo ATP synthesis.

It is concluded that the data obtained are in agreement with the concept of the energy storing by the Na+ and K+ gradients. Other functions of these gradients and the mechanisms of their formation are discussed.
Kinetic Evidence for an Activation Step Following Binding of Human Interferonα2 to the Membrane Receptors of Daudi Cells
Authors: MOGENSEN Knud E. , BANDU Marie‐Thérèse
Published: March 2005

A single species of human interferon α (IFN] was labelled with 125I to high incorporation for binding studies on the B‐lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination.

Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same intial culture of cells. 125I‐labelled IFNα (IFNα prepared from Escherichia coli carrying human α2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 ± 0.07 days−1) at a mean concentration of 235000 ± 20000 cells ml−1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibirum some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the ‘steady‐state’, appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occuring consecutively over the wholw of the dose range studied (1 – 100 U/ml; 0.25–25pM IFN).

In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double‐reciprocal plot (i.e. in analogy to Michaelis‐Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 PM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited.

We propose that the first reaction represents binding of IFN to surfac receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.
Investigation by Photochemically‐Induced Dynamic Nuclear Polarization and Nuclear Overhauser Enhancement 1H‐NMR of the Interaction between β‐Endorphin and Phospholipid Micelles
Authors: ZETTA Lucia , HORE Peter J. , KAPTEIN Robert
Published: March 2005

The photochemically‐induced dynamic nuclear polarization technique has been used to investigate the access of a photoexcited flavin dye to tyrosyl and histidyl residues in [Met]enkephalin and human and camel β‐endorphins, both alone and in the presence of n‐dodecylphosphorylcholine micelles. The results indicate that the mode of binding of Tyr‐1, but not of residue 27, is similar in the two endorphins and differs from that of Tyr‐1 in [Met]enkephalin. In human β‐endorphin, accessibility and mobility of Tyr‐27 are strongly reduced in the presence of lipid at physiological pH, whereas in camel β‐endorphin His‐27 becomes becomes immobilized only at high pH. Moreover, nuclear Overhauser enhancement experiments suggest a rigidifying influence of the peptide on the polar head groups of the micelles.
Regulation of Ca2+Efflux in Rat Liver Mitochondria
Authors: BERNARDI Paolo , AZZONE Giovanni Felice
Published: March 2005

The paper analyzes the relationship between membrane potential (ΔΨ), steady state pCa0(‐log [Ca2+] in the outer aqueous phase) and rate of ruthenium‐red‐induced Ca2+efflux in liver mitochondria. Energized liver mitochondria maintain a pCa0of about 6.0 in the presence of 1.5 mM Mg2+and 0.5 mM Pi. A slight depression ofΔΨresults in net Ca2+uptake leading to an increased steady state pCa0. On the other hand, a more marked depression ofΔΨresults in net Ca2+efflux, leading to a decreased steady‐state pCa0. These results reflect a biphasic relationship betweenΔΨand pCa0, in that pCa0increases ofΔΨup to a value of about 130 mV, whereas a further increase ofΔΨabove 130 mV results in a decrease of pCa0. The phenomenon of Ca2+uptake following a depression ofΔΨis independent of the tool used to affectΔΨwhether by inward K+current via valinomycin, or by inward H+current current through protonophores or through, F1‐ATP synthase, or by restriction of e−flow.

The pathway for Ca2+efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCa0at steady state and by an increased rate of ruthenium‐red‐induced Ca2+efflux. By restricting the rate of e−flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium‐red‐induced Ca2+efflux on the value ofΔΨis observed, in that the rate of Ca2+efflux increases with the value ofΔΨ. The pCa0is linearly related to the rate of Ca2+efflux.

Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP‐supplemented mitochondria, is followed by a phase of Ca2+uptake, which is reversed by atractyloside.

These findings support the view that Ca2+efflux in steady state mitochondria occurs through an independent,ΔΨ‐controlled pathway and that changes ofΔΨduring oxidative phosphorylation can effectively modulate mitochondrial Ca2+distribution by inhibiting or activating theΔΨ‐controlled Ca2+efflux pathway.
The Nature of the Electron Spin Resonance Signal during Aerobic Uptake of Mn2+ in Mitochondria from Rat Liver
Authors: BRAGADIN Marco , POZZAN Tullio , AZZONE Giovanni Felice
Published: March 2005

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high‐energy membrane sites or to complexes of Mn2+ with inorganic phosphate.

Identification of the spin exchange signal with a Mn‐Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N‐ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Piis generated in the matrix, in the presence of N‐ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal.

The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50–60 nmol × mg protein −1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations.

The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93–99] that the spin exchange singal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.
Reconstitution of a Functional β‐Adrenergic Receptor using Cholate and a Novel Method for Its Functional Assay
Authors: GAL Alma , BRAUN Sergei , FEDER Debra , LEVITZKI Alexander
Published: March 2005

Solubilization of purified turkey erythrocyte membranes at increasing cholate to protein ratios and in the presence of salt, extracts up to 20% of the β‐adrenergic receptor together with the GTP stimulatory protein (Ns) of adenylate cyclase. Upon removal of the cholate, by active absorption on Bio‐beads, the functional interaction between the β‐receptor and the GTP regulatory protein Ns is quantitatively restored. The receptor (R) in the presence of l‐isoproterenol and p[NH]ppG is able to catalyze the activation of Ns to its permanently active state, N's p[NH]ppG, with a rate constant (kon) identical to that of the native membrane. Reconstitution of the R/Ns mixture using poly(ethyleneglycol)‐6000 restores the receptor binding properties as effectively as SM‐2 Bio‐beads. Unlike SM‐2 Bio‐beads, however, poly(ethyleneglycol) is not as efficient in restoring the R to Ns functional coupling. In this communication we also report on the ability to monitor quantitatively N's. p[NH]ppG, using native turkey erythrocyte membranes in the presence of Lubrol‐PX as the source of the catalytic unit (c) of adenylate cyclase. The latter method is as efficient as using S49 AC‐ lymphoma cell membranes but much less expensive. Using this technique, we also demonstrate that when the Ns to C interaction is nullified, employing treatment with N‐ethylmaleimide, the parameters which characterize R to Ns coupling remain unchanged.
Antigenic determinants of human placental and testicular placental‐like alkaline phosphatases as mapped by monoclonal antibodies
Authors: MILLÁN José Luis , STIGBRAND Torgny
Published: March 2005

Placental alkaline phosphatase (PLAP) in humans shows a high degree of genetic polymorphism as disclosed by electrophoretic analysis. Human testes contain trace amounts of a PLAP‐like enzyme, that although immunologically cross‐reactive with PLAP, shows unique catalytic properties.

As an alternative approach to study enzyme polymorphism we have developed monoclonal antibodies to purified allelic variants of PLAP. Five different monoclonal antibodies are described in this report. The antibodies react with different epitopes on the PLAP molecule. Both conformational dependent and independent determinants are detected. Two epitopes are modified when comparing the S and F allelic variants of PLAP. One epitope is common to PLAP and the intestinal isoenzyme of alkaline phosphatase. The five epitopes appear to be mapped on two rather distant antigenic domains. Combinations of any two antibodies binding to different domains give immunoprecipitates with PLAP on Ouchterlony tests and give good response in sandwich enzyme‐linked immunosorbent assays.

A study of PLAP‐like enzyme in 32 individual testis samples indicates differences in four of the epitopes when compared with PLAP. Four types of testicular enzymes can be distinguished based on their reactivities. These results indicate structural differences between the testicular PLAP‐like enzyme and PLAP. These differences are compatible with an underlying genetic mechanism.