bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Some Properties of Tyrosine 3‐Monooxygenase from Rat Adrenal
Authors: OKUNO Sachiko , FUJISAWA Hitoshi
Published: March 2005

Tyrosine 3‐monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1600 nmol min−1 mg protein−1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross‐linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein.

Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2‐amino‐4‐ hydroxy‐6‐methyl‐5,6,7,8‐tetrahydropteridine decreased from 220 μM to 58 μM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phos‐ phatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.
Phospholipid Interconversions in Mycoplasma capriculum
Authors: GROSS Zvi , ROTTEM Shlomo , BITTMAN Robert
Published: March 2005

Mycoplusma cupricolurn cells increase their phospholipid content by incorporating exogenous phospholipids from the growth medium. Growing the cells in media with increasing serum concentrations resulted in a massive incorporation of phosphatidylcholine and sphingomyelin (up to about 50% of total phospholipids) into the cell membrane. The incorporation of the exogenous phospholipids had essentially no effect on the rate of cell growth and did not decrease the overall phospholipid biosynthesis of the cells. Thus, the ratio of phospholipid to protein in membranes from cells grown with 5% horse serum was 0.5 (μmol/mg) compared to 0.3 (μmol/mg) in cells grown without serum, and the relative content of charged polar lipids was apparently decreased. The consequence of the incorporation of exogenous phosphatidylcholine was an alteration in the relative amount of the major end‐products of the de novo phospholipid biosynthesis; a marked increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol was observed. The possibility that the increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol is part of a control mechanism to maintain a mixture of bilayer and non‐bilayer lipids is discussed.
Purification and Characterization of Two Forms of Rat Plasma Proangiotensin
Authors: VOIGT Jürgen , WITTMANN‐LIEBOLD Brigitte , KÖSTER Hubert
Published: March 2005

Two forms of rat plasma proangiotensin were purified by (NH4)2SO4 fractionation, chromatography on DEAE‐cellulose at pH 6.5, DEAE‐Sepharose at pH 8.9, Sephadex G‐150, hydroxyapatite and hexyl‐agarose. Both forms were finally separated by affinity chromatography on concanavalin‐A ‐ Sepharose. Presence or absence of carbohydrate side chains seems to be the only difference between these forms of proangiotensin. Both proteins consist of single polypeptide chains having apparent molecular weights of 52000 and 55000 and isoelectric points around 4.7 and 4.4, respectively. No significant difference between the proteins could be observed with respect to the amino‐terminal amino acid sequence which was found to be the same (H2N‐Asp‐ Arg‐Val) as for angiotensin I and II. Furthermore, extensive digestion with renin, releasing the decapeptide angiotensin I, did not significantly reduce the molecular weights of both polypeptides. It can therefore be concluded that the angiotensin I peptide is located at the amino terminus of the prohormone. Kinetic constants measured for the release of angiotensin I by renin were found to be Km= 5.0 μM proangiotensin and V = 270 nmol of angiotensin I h−1 unit renin−1 for the concanavalin‐A‐binding form and Km= 5.6 pM proangiotensin and V = 250 nmol angiotensin I h−l unit renin−1 for the prohormone which did not bind to concanavalin‐A‐Se‐ pharose. The form of proangiotensin not bound to concanavalin‐A‐Sepharose was found to be more thermally labile (tm of 59.0°C) than the form binding to concanavalin A (tm of 61.5°C, where tm temperature at which 50% reactivity is lost).
Rate of Elongation of Polyphenylalanine in vitro
Authors: WAGNER E. Gerhart H. , JELENC Pierre C. , EHRENBERG Måans , KURLAND Charles G.
Published: March 2005

Ribosomes purified from Escherrchipa coli were preincubated with AcPhe‐tRNA and poly(U). Then purified components necessary for polypeptide synthesis were added. Incubation of the complete system led to a burst of elongation which lasted for nearly 10 s. During the initial burst approximately 10% of the ribosomes participated in the elongation of poly(Phe) at an average rate per ribosome close to eight peptide bonds/s. The missense error rate with leucine was 4x10−4 during the burst. Accordingly, the preincubated elongation system functions at a rate, as well as an accuracy, close to those of protein synthesis in vivo.
Effect of Secondary Structure of Messenger Ribonucleic Acid on the Formation of lnitiation Complexes with Prokaryotic and Eukaryotic Ribosomes
Authors: ZAGÓRSKA Ludwika , CHROBOCZEK Jadwiga , KLITA Stefan , SZAFRAŃSKI Przemystaw
Published: March 2005

The effect of modification of the secondary structure of phage f2 RNA and brome mosaic virus (BMV) RNA 3 on the formation of initiation complexes in Escherichia coli and wheat germ protein‐synthesizing systems was studied. Modification of the RNAs was achieved by using O‐methylhydroxylamine, which specifically reacts with cytosines; this leaves the initiation codons unchanged and, under denaturing conditions, leads to irreversible unfolding of the RNA.

E. coli ribosomes interact with newly exposed AUG/GUG codons in the modified templates forming polysomes, whereas they form monosomes with native f2 RNA or BMV RNA 3. With wheat germ ribosomes, disomes are formed in the presence of BMV RNA 3, either native or modified. With f2 RNA, eukaryotic ribosomes form monosomes, independent of the secondary structure of the template.

The results indicate that, in contrast to prokaryotic ribosomes, binding of eukaryotic ribosomes to f2 RNA or BMV RNA 3 is not affected by modification of the secondary structure of these messengers.
Demonstration of a Stable Occluded Form of Ca2+ by the Use of the Chromium Complex of ATP in the Ca2+‐ATPase of Sarcoplasmic Reticulum
Authors: SERPERSU Engin Halit , KIRCH Ulrïke , SCHONER Wilhelm
Published: March 2005

Sarcoplasmic reticulum Ca2+‐ATPase is slowly inactivated by the β, γ‐bidentate of the chromium(III) complex of ATP (CrATP). The rate of inactivation depends on the presence of calcium ions in the incubation medium. Half‐maximal inactivation rate is reached at 80 nm Ca2+. The dissociation constant of enzyme‐CrATP complex at 26°C is 31 μM. Inactivation of the enzyme by CrATP is protected competitively by ATP. From this a dissociation constant of the enzyme ATP complex of 6.2 μM is calculated.

The inactivation of Ca2+‐ATPase is proportional to the incorporation of 32P from [γ‐32P]CrATP into the 100000‐Mr protein. Concomitant with the inactivation there is also an incorporation of 45Ca2+ into the purified Ca2+‐ATPase. A ratio of 45Ca2+ binding to phosphorylation of 1.7‐1.9 is calculated from the capacities of both processes as well as from their kinetics.

When Ca2+ uptake into sarcoplasmic reticulum is fuelled by ATP, treatment of the vesicles with the Ca2+ ionophore X‐537‐A or the production of leaks by adding deoxycholate leads to a loss of already accumulated 45Ca2+. Such treatment, however, leaves the 45Ca2+ in the vesicles of Ca2+‐ATPase unaffected, if ATP had been replaced by CrATP during the Ca2+ uptake. It is possible to localize 45Ca2+ together with the phosphointermediate in the dodecylsulfate electrophoresis on the 100000‐Mr protein and to a minor degree on the 200000‐Mr dimer. This apparently occluded 45Ca2+ is rapidly lost by treatment of the enzyme with trichloroacetic acid.

It is concluded, that CrATP acts as an ATP analogue on Ca2+‐ATPase of sarcoplasmic reticulum. Hydrolysis of CrATP, as in (Na++ K+)‐ATPase [Pauls, H., Bresenbröcker, B., and Schoner, W. (1980) Eur. J. Biochem. 109, 523‐533] leads to a stable phosphointermediate, which appears to bind 45Ca2+ in a stable occluded form. Both processes apparently lead to the suicide of the Ca2+‐ATPase.
Similarities between Mouse and Rat‐Liver Microsomal Cytochromes P‐450 Induced by 3‐Methylcholanthrene
Authors: CHEN Yuan‐Tsong , LANG Matti A. , JENSEN Nancy M. , NEGISHI Masahiko , TUKEY Robert H. , SIDRANSKY Ellen , GUENTHER Thomas M. , NEBERT Daniel W.
Published: March 2005

Anti‐(P1‐450) and anti‐(P‐448) are two antibodies developed against distinctly different forms of 3‐methyl‐ cholanthrene‐induced cytochrome P‐450 in C57BL/6N mouse liver microsomes [Negishi and Nebert, J. Biol. Chem. 254, 11015‐11023 (1979)]. The effects of these antibodies on Long‐Evans and Sprague‐Dawley rat liver microsomes and rat ‘minimal deviation’ hepatoma 7777 microsomes were studied.

Rat microsomal aryl hydrocarbon hydroxylase activity, induced by polycyclic aromatic compounds, is prefer‐ entially inhibited by anti‐(P1‐450) more so than anti‐(P‐448). Polycyclic‐aromatic‐induced rat liver microsomal acetanilide 4‐hydroxylase activity is preferentially inhibited by anti‐(P‐448) more so than anti‐(P1‐450). Anti (P1‐450) precipitates a 56‐kDa moiety in polycyclic‐aromatic‐induced rat liver microsomes. Anti‐(P‐448) precipitates a 55‐kDa moiety in control, phenobarbital‐treated, or polycyclic‐aromatic‐induced rat liver microsomes. A heterogeneity was found amongst individual Morris ‘minimal deviation’ hepatomas 7777, indicating the 3‐methylcholanthrene‐induced aryl hydrocarbon hydroxylase and acetanilide 4‐hydroxylase'activities must represent different forms of rat P‐450.

Clone 46 DNA, a portion of the mouse cloned P1‐450 structural gene, hybridizes to one gene (or a small number of genes) in rat liver and to 3‐methylcholanthrene‐induced rat 23‐S P1‐450 mRNA. As has already been shown in the mouse [Tukey, Nebert and Negishi, J. Biol. Chem. 256, 6969‐6974 (1981)], rat liver P1‐450 induction is under transcriptional control, and there is no evidence for gene amplification or some gross form of DNA rearrangement.

These data demonstrate striking similarities between rat and mouse for the P1‐450 structural gene, the P1‐450 mRNA, the P1‐450 protein and the P‐448 protein. Polycyclic‐aromatic‐induced P1‐450, and its association with polycyclic‐aromatic‐induced aryl hydrocarbon hydroxylase activity, therefore does not appear to be closely correlated with polycyclic‐aromatic‐induced P‐448 in either rat or mouse.
Identity Problems Concerning Subunits of the Membrane Factor of the Mitochondria1 ATPase of Saccharomyces cerevisiae
Authors: SOMLO Marie , COSSON Jacky , CLAVILIER Lea , KRUPA Mireille , LAPORTE Iréne
Published: March 2005

As confirmed by fingerprint analysis, high‐resolution acrylamide gel electrophoresis identifies subunits of the mitochondrial ATPase complex directly at the level of Coomassie‐blue‐stained yeast mitochondria. Using this technique, the following results were obtained.

Three classes of subunits have been found in the ATPase complex. (a) The classical F1 peptides of cytoribosomal origin (57, 53 and 30.5 kDa). (b) Two peptides of mitoribosomal origin with high cycloheximide‐ resistant label: one of 7.8 kDa (the OLI1 gene product), the second of 20 kDa (the OLI2 gene product). Neither of these peptides is readily stained by Coomassie blue in the purified ATPase complex. (c) Four Coomassie‐blue‐stained peptides (27.5, 25, 23.5 and 22.5 kDa), the last one being subunit 6, which (like class b) functionally qualify as subunits of the membrane factor of the ATPase complex. As shown by labeling with 35SO2−4 and 3H‐labeled amino acids, their biosynthesis is only very slightly cycloheximide‐resistant, although it is submitted to coordinate regulation distinct from that of the class F1 peptides.

Consequently it was shown that the OLI2 gene product, a 20‐kDa peptide with high cycloheximide‐ resistant label, which was generally taken to be ‘subunit 6’ of the ATPase, is not in fact identical to this peptide.
Studies on the Biosynthesis of Microsomal Membrane Proteins
Authors: OKADA Yoshiie , FREY Alan B. , GUENTHNER Thomas M. , OESCH Franz , SABATINI David D. , KREIBICH Gert
Published: March 2005

The site of synthesis and mechanism of insertion into membranes of several microsomal polypeptides was studied using translation system programmed in vitro with polysomes or with mRNA extracted from free and membrane‐bound rat liver polysomes. Primary translation products of cytochrome b5, NADH: cytochrome b5 oxidoreductase, NADPH: cytochrome P‐450 oxidoreductase and epoxide hydrolase were isolated by specific immunoprecipitation and compared with the mature proteins. The following observations were made:

While cytochrome b5 and NADH: cytochrome b5 oxidoreductase are synthesized in free polysomes, NADPH: cytochrome P‐450 oxidoreductase and epoxide hydrolase are made in membrane‐bound polysomes.

In all cases the radioactively labelled products synthesized in vitro comigrated with the purified native polypeptides. Since these proteins are not glycoproteins, it can be inferred that in vivo they do not undergo proteolytic cleavage during or after translation.

Levels of translatable mRNA coding for NADPH: cytochrome P‐450 oxidoreductase or epoxide hydrolase were increased by phenobarbital or by various carcinogens respectively.

A comparison of amino‐terminal segments of the native epoxide hydrolase and that synthesized in vitro showed that this protein retains an amino‐terminal polypeptide sequence which resembles the transient insertion signal found in presecretory proteins.

The amino‐terminal region of NADPH: cytochrome P‐450 oxidoreductase contains 7 leucine residues in its first 15 amino acids and can, therefore, be presumed to be the hydrophobic portion which anchors this poly‐ peptide to the membrane.

Mechanisms which may account for the spatial disposition of the membrane proteins and for their selective accumulation in endoplasmic reticulum membranes are discussed.
Investigation of the Iron‐Sulfur Clusters in Some Mitochondria1 Mutants of Saccharomyces cerevisiae
Authors: CAPEILLERE‐BLANDIN Chantal , OHNISHI Tomoko
Published: March 2005

We have undertaken a systematic analysis of the iron‐sulfur clusters present in strains of Saccharomyces cerevisiae carrying mutations in the COB‐BOX region of the mitochondrial DNA coding for cytochrome b, and in OXI loci of the mitochondrial DNA coding for subunits of cytochrome oxidase. The iron‐sulfur clusters and cytochromes in complexes II and III of the yeast mitochondrial respiratory chain have been largely identified. Mutant phenotypes are defined in terms of succinate dehydrogenase and cytochrome c reductase activities, low‐ temperature absorption spectra and electron paramagnetic resonance spectra.

From quantitative comparison of iron‐sulfur clusters in mitochondrial preparations of BOX, OXI and rho° mutants, we have made the following two sets of observations. (a) Among the components of complexIII, the appearance of Rieske's iron‐sulfur cluster seems to be correlated only to the presence or absence of the cytochrome b; moreover Rieske's cluster is absent even when polypeptide fragments of apocytochrome b are known to be synthesized. This observation constitutes a very interesting example illustrating perturbation of the incorporation of a cytoplasmically made protein (Rieske's protein) by structural alterations of a mitochondrially made protein (cytochrome b), both being assembled within the same multiprotein complex. The situation differs with another cytoplasmically‐made protein (cytochrome c1). (b) Clusters S‐1, S‐2 and S‐3 associated with complex II are present, irrespective of the type of mutation, at about ∼ 10‐20 % of the level of that in the wildtype species; this partial repression could be a result of the respiratory deficiency of these mutants.
Limited Proteolysis and Proton NMR Spectroscopy of Bacillus stearothermophilusPyruvate Dehydrogenase Multienzyme Complex
Authors: DUCKWORTH Harry W. , JAENICKE Rainer , PERHAM Richard N. , WILKIE Andrew O. M. , FINCH John T. , ROBERTS Gordon C. K.
Published: March 2005

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0°C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent M, fell from 57000 to 45000 as judged by sodium dodecylsuphate/poly‐ acrylamide gel electrophoresis. The inactive chymotrypsin‐treated enzyme had lost the lipoic‐acid‐con‐ taining regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0°C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28000 and was accompanied by disassembly of the complex. The lipoic‐acid‐containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes.

Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic‐acid‐containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active‐site coupling in the 2‐oxo acid dehydrvgenase multienzymc complexes.
An Extracellular Poly(3‐Hydroxybutyrate) Depolymerase from Alcaligenes faecalis
Authors: TANIO Tomoharu , FUKUI Tetsuya , SHIRAKURA Yuri , SAITO Terumi , TOMITA Kenkichi , KAIHO Tatsuo , MASAMUNE Satoru
Published: March 2005

A strain of Alcaligenes faecalis T1, which was isolated from activated sludge, excreted an extracellular poly(3‐hydroxybutyrate) depolymerase as it grew in a medium containing poly(3‐hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48000 as determined by Sephadex G‐100 filtration, and 50000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3‐hydroxybutyrate) purified from Zoogloea ramigeva 1–16‐M, but did not attack the bacterial native poly(3‐hydroxybutyrate containing granules. K, values were 13.3 μpg/ml(= 0.78 μM, based on an estimated average molecular weight of 17000) for poly(3‐hydroxybutyrate) and 5.4mM for the trimeric ester of D(–)‐3‐hydroxybutyric acid. Analysis of hydrolytic products of poly(3‐hydroxybutyrate), several oligomeric esters of D(–)‐3‐hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing D(‐)‐3‐hydroxybutyrate dimer units one at a time.
Immunoglobulin G Biosynthesis in a Human Lymphoblastoid Cell Line
Authors: OWEN Michael J. , KISSONERGHIS Anne‐Maija
Published: March 2005

The cultured human B lymphoblastoid cell line Maja synthesises two forms of the ‐γ heavy chain of immunoglobulin G (IgG) that differ in apparent molecular weight. The lower‐molecular‐weight form is secreted into the culture medium as a water‐soluble product in association with light chains and comigrates on dodecyl sulphate polyacrylamide gels with serum IgG γ chains. The higher‐molecular‐weight form is not detected in culture supernatants. In distinction to the secreted form, the higher‐molecular‐weight form is labelled by a lipophilic, photoactivatable nitrene and is inserted asymmetrically in a transmembrane orientation into rough microsomes. It is concluded that Maja cells synthesise secretory (γs) and membrane‐associated (γm) forms of IgG heavy chains. Both forms of the heavy chain are glycosylated, and can contain one or two asparagine‐linked glycan units. The γm and γs heavy chains differ by about 10000 in apparent molecular weight. This difference resides exclusively in the polypeptide moiety. Although part of the difference comprises a transmembrane peptide and a cytoplasmic tail of apparent molecular weight about 2000 for γm chains, a substantial segment of unique peptide is most probably present on the non‐cytoplasmic side of the bilayer. The ionophore monensin inhibits the intracellular transport of γs and γm chains at a stage when they are sensitive to the enzyme endo‐β‐N‐acetylglucosaminidase H. In contrast, HLA‐A and HLA‐B antigens reach a stage at which they are insensitive to this enzyme in the presence of monensin, although their surface expression is inhibited by the ionophore. The implications of these results for the intracellular transport of membrane‐associated glycoproteins are discussed.
Activity of Carbamoyl‐Phosphate Synthetase (Ammonia) in Isolated Rat‐Liver Mitochondria: Cycling of Carbamoyl Phosphate in the Absence of Ornithine
Authors: LOF Cor , WANDERS Ron J. A. , MEIJER Alfred J.
Published: March 2005

When NH3was added to isolated rat‐liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate.

Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mito‐ chondria had been preincubated with NH3and unlabelled bicarbonate, revealed that flux through carbamoyl‐phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated.

It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non‐enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl‐phosphate synthetase.
Glucocorticoid Hormones Have a Permissive Role in the Phosphorylation of L‐Type Pyruvate Kinase by Glucagon
Authors: POSTLE Anthony D. , BLOXHAM David P.
Published: March 2005

The incorporation of [32P]phosphate into L‐type pyruvate kinase in response to glucagon was studied in hepatocytes isolated from control and adrenalectomized rats. In control cells, pyruvate kinase phosphorylation was maximally stimulated by 210% by glucagon. Adrenalectomy reduced both the basal extent of phosphorylation and the response to glucagon to 25% of those measured in control hepatocytes. This impaired pyruvate kinase phosphorylation was associated with altered kinetic properties of L‐type pyruvate kinase. Glucagon injected in vivo (1 mg/kg) into adrenalectomized rats did not increase the substrate co‐operativity of L‐type pyruvate kinase. These results suggest a molecular basis for the well‐recognised glucocorticoid hormone‐dependence for the stimulation by glucagon of hepatic gluconeogenesis. Further effects of this defective enzyme regulation on hepatic metabolism are suggested by the observation that adrenalectomy abolished the ability of glucagon to inhibit hepatic fatty acid biosynthesis both in vivo and in isolated hepatocytes.
Synthesis and Characterization of Neurotensin Analogues for Structure/Activity Relationship Studies
Authors: GRANIER Claude , VAN RIETSCHOTEN Jurphaas , KITABGI Patrick , POUSTIS Claudine , FREYCHET Pierre
Published: March 2005

Neurotensin and several sequence analogues have been synthesized using solid‐phase technology. The purity of the following derivatives: neurotensin, neurotensin‐(10–13), neurotensin‐(9–13). neurotensin‐(8–13), neurotensin‐(6–13), neurotensin‐(4–13), [Cit8]neurotensin‐(8–13), [Lys8]neurotensin‐(8–13), [Cit9]neurotensin‐(8–13),[Lys9]neurotensin‐(8–13), [Phe11]neurotensin‐(8–13), [Ala12]neurotensin‐(8–13) and [Ala13]‐ neurotensin‐(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse‐phase high‐ performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N‐acetylation of neurotensin‐(6–13), neurotensin‐(8–13), [Cit8]neurotensin‐(8–13), [Cit9]‐ neurotensin‐(8–13), [Lys8]neurotensin‐(8–13), [Lys9]neurotensin‐(8–13) and [Phe11]neurotensin‐(8–13), as well as native xenopsin, were all tested for binding competition with [3H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C‐terminal region analogues up to the size of neurotensin‐ (8–13). The two peptides neurotensin‐(6‐ 13) and neurotensin‐(4–13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C‐terminal peptide ‐Arg‐Arg‐Pro‐Tyr‐Ile‐Leu fulfills all the structural requirements for mimicking the entire sequence, provided its α‐amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin‐(8–13), acts similarly to acetyl‐neurotensin‐(8–13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C‐terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.
Two Identical Subunits of the EcoRI Restriction Endonuclease Co‐operate in the Binding and Cleavage of the Palindromic Substrate
Authors: ALVES Jürgen , PINGOUD Alfred , LANGOWSKI Jörg , URBANKE Claus , MAASS Günter
Published: March 2005

The cleavage of radioactively labelled double‐stranded d(G‐G‐A‐A‐T‐T‐C‐C) was studied in single turnover experiments with substrate and enzyme both being in the micromolar range. The reaction rate was found to increase with enzyme concentration until a ratio of one tetrameric enzyme to two double‐stranded substrates was reached, further increase of the enzyme concentration then leads to a sharp decline of the reaction rate. These finding are interpreted in the following manner. (a) Two subunits of the EcoRI endonuclease co‐operate in binding and possibly also in cleaving the palindromic substrate. (b) The enzymatic action of the EcoRI endonuclease is inhibited by excess enzyme, possibly due to unspecific binding of the enzyme‐substrate complex. The sell‐inhibition of EcoRI endonuclease has also been observed with macromolecular substrates.
Influence of the Structure of Water on the Hydrolysis of Cytidine 2′,3′‐Phosphate Catalysed by Bovine Pancreatic Ribonuclease A
Authors: BIOSCA Josep A. , TRAVERS Franck , CUCHILLO Claudi M.
Published: March 2005

The study of the temperature dependence of the hydrolysis of cytidine 2′,3′‐phosphate by bovine pancreatic ribonuclease A (EC 3.1.27.5) at pH 7.0 by using the pH‐stat method showed a transition at 4 C [J. A. Biosca and C. M. Cuchillo (1980) Biochem. J. 189, 655–6571, The breaks found in the Van't Hoff and Arrhenius plots at pH 7.0 were confirmed in the present work by following the reaction spectrophotometrically with a stopped‐flow spectrophotometer adapted to the use of sub‐zero temperatures. Similar results were found at pH 5.5. In addition it was found that the discontinuity disappears when 40% ethyleneglycol is present in the reaction mixture. However, in this latter instance a discontinuity around 0°C appears in the Arrhenius plot. To explore the possibility that all these effects were due to a conformational transition in the protein, thermal perturbation experiments were carried out with the enzyme. A change in the slope of the plot of as a function of temperature was found around 6°C at pH 7.0 but not at pH 5.5. The results reported here can be interpreted as due to a change in the protein structure induced by the change of the structure of water. The studies carried out in the presence of ethyleneglycol also open the way to the cryoenzymological experimentation on ribonuclease.
Structural Studies on Rat Prostatic Binding Protein
Authors: PEETERS Ben , MOUS Jan , ROMBAUTS Wilfried , HEYNS Walter
Published: March 2005

The amino acid sequence of component C1, the polypeptide specific for subunit F of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the intact protein and on the most relevant fragments isolated from trypsin, chymotrypsin, thermolysin and Staphylococcus aureus protease digests of the 14C‐labelled S‐carboxamidornethylated component C1.

Component C1 contains 88 amino acids corresponding to a molecular weight of 10246. It is an acidic polypeptide due to the presence of 17 acidic residues; its thrce cysteine residues are almost symmetrically distributed over the peptide chain. Highly polar regions are found in positions 17‐27 and 37‐47, while the C‐terminal part of the molecule contains two hydrophobic segments
Protease Resistance and Conformation of Laminin
Authors: OTT Ursula , ODERMATT Erich , ENGEL Jürgeii , FURTHMAYR Heinz , TIMPL Rupert
Published: March 2005

Laminin, obtained from a tumor basement membrane, was treated with neutral proteases (trypsin, chymotrypsin, elastase, subtilisin, Stuphylococcus aureus protease), which all produced similar fragment patterns upon prolonged digestion. The patterns were different from those obtained for fibronectin, which showed a much higher susceptibility to proteolysis by the same enzymes. Four large fragments could be purified which accounted together for more than half of the mass of laminin. They were found to differ in size, amino acid composition, spectral properties and antigenicity. The largest fragment 1 (Mr 260000‐300000) was rich in half‐cystine (120 residues/1000) and showed a circular dichroism spectrum indicating the absence of α helix and β structure. Fragment 3 (Mr= 50000) possessed beta; structure and was able to bind onto heparin‐Sepharose. Fragments 2 (Mr= 50000) and 4 (Mr= 75000) were related structures and their relative yields depended on the protease used. They showed spectra similar to those of fragment 1. Electron microscopy revealed that fragment 1 consists of three rodlike elements (length 26 nm) connected to each other at one end. The other fragments appeared as globules (fragment 3), short rods (fragment 2) or globules connected to a short rod (fragment 4). Data obtained from limited proteolysis indicated that fragment 1 and 4 (or 2) are in close proximity to each other in the three short arms of laminin, which in its intact form has the shape of an asymmetric cross. The long arm appeared to be readily degraded by proteases.

Circular dichroism studies of native laminin indicated about 55%, aperiodic structures, 15%β structure and 30%× helix. The α helix was readily destroyed by proteolysis and showed a sharp, reversible transition at 58° C. Stability of these structures was decreased by reduction of disulfide bonds or by increasing concentrations of guanidine. Heat denaturation rendered laminin susceptible to plasmin, which did not degrade the native protein. Cleavage occurred mainly within the 440000‐Mrpolypeptide chain of laminin and was accompanied by a partial loss of the long arm.
Glucose Transport in Salmonella typhimurium and Escherichia coli
Authors: POSTMA Pieter W. , VAN REE Ronald , NEYSSEL Oense M.
Published: March 2005

We have investigated the claim by Schweiger and coworkers [Eur. J. Biochem. I02 (1979) 231 ‐236] that glucose transport in Escherichia coli is catalyzed mainly by an ATP‐dependent transport system instead of the phosphoenolpyruvate : sugar phosphotransferase system. A major argument was the differential effect of 2.4‐dinitrophenol on glucose uptake and the transport of its non‐metabolizable analogue, methyl α‐glucoside. Whereas the first was inhibited, the second was stimulated. When subsequent glucose metabolism is prevented by introducing mutations that eliminate glucose 6‐phosphate metabolism, 2,4‐dinitrophenol does not inhibit glucose transport. Although dinitrophenol inhibited in wild‐type cells of E. coli and Salmonella typhimurium the uptake of I4C label in cells using [U‐14C]glucose as a substrate, disappearance of glucose from the medium was not affected or only slightly affected. Since uptake represents a combination of transport and subsequent metabolism, retention of labelled material depends on the balance of incorporation of label in cellular material and efflux of labelled compounds. Our experiments show that inhibition of the uptake of labelled glucose by 2,4‐dinitrophenol is not due to inhibition of transport as suggested by Schweiger and coworkers, but to increased efflux of labelled compounds such as acetate and pyruvate. In addition, incorporation of label in cellular material is lowered by dinitrophenol. Inhibition of uptake by dinitrophenol is found with many labelled sugars, including mannitol, galactose and glycerol, the transport of which is energized in quite different ways.

We conclude that there is no need to postulate a novel ATP‐driven system for glucose transport. All results can be explained with the phosphoenolpyruvate : glucose phosphotransferase system as the main if not sole glucose transport system.
Pregnenolone Efflux from Mitochondria of Bovine Adrenal Cortex
Authors: SHEARS Stephen B. , BOYD George S.
Published: March 2005

Pregnenolone efflux from bovine adrenal cortex mitochondria was measured with the aid of rapid separation of the organelles from the incubation by centrifugation through silicon oil. In the absence of bovine serum albumin or cytosolic supernatant (100000 × g), an equilibrium distribution of newly synthesized pregnenolone was attained after 2 min at 30°C; 20% of total pregnenolone was located outside the mitochondria. The presence of 13 mg albumin/ml or 13 mg supernatant protein/ml increased the exogenous proportion of pregnenolone to over 90% and 75% respectively without affecting the rate of cholesterol side‐chain cleavage. The binding of pregnenolone by both albumin and supernatant was non‐specific and non‐saturable. It is suggested that in vivo the mitochondrial membranes do not present a significant barrier to pregnenolone efflux from the organelle.
The Segregation into Microsomal Vesicles and Core‐Glycosylation in vitro of a 300‐kDa Rat Thyroglobulin Subunit
Authors: ALVINO Claudio G. , TASSI Vittorio , POLISTINA Claudio , DILAURO Roberto , BONATTI Stelano
Published: March 2005

Translation of rat thyroid mRNA in a cell‐free protein synthesis system derived from rabbit reticulocytes results in synthesis of a 300‐kDa thyroglobulin polypeptide [C. G. Alvino et al. (1982) >FEBS Lett. 137, 307 – 313]. In the presence of dog pancreas microsomal membranes this polypeptide is segregated into the microsomal vesicles and core‐glycosylated, as shown by increased protection against proteolytic treatment and binding to concanavalin‐A ‐ Sepharose. The segregation process appears to be strictly coupled to glycosylation, i.e. no detectable amounts of unglycosylated chains are found in the vesicles, and both processes only occur cotranslationally. The glycosylated protein exhibits lower electrophoretic mobility with respect to its non‐glycosylated form and comi‐ grates with a glycosylated 330‐kDa polypeptide found in rat thyroid epithelial cells cultured in vitro [F. S. Ambesi‐ Impiombato et al. (1980) Proc, Nat1 Acad. Sci. USA, 77, 3455‐3459]. Both glycosylated and non‐glycosylated forms are immunoprecipitated by antibodies to 19‐S rat thyroglobulin. Some features of the tyroglobulin poly‐ peptide segregation and glycosylation have been studied by synchronized translation of rat thyroid mRNA : (a) the signal sequence seems to be located at the amino‐terminal portion of the nascent polypeptide chain; (b) the distribution of the carbohydrate units is mostly clustered in the amino‐terminal half of the protein as monitored by posttranslational analysis of the polypeptides synthesized in the presence of pancreatic membranes during competition experiments carried out with the detergent Nonidet P40.
N‐Terminal Amino Acid Sequence of Sialoglycoprotein D (Glycophorin C) from Human Erythrocyte Membranes
Authors: DAHR Wolfgang , BEYREUTHER Konrad , KORDOWICZ Maria , KRÜUGER Jürgen
Published: March 2005

The amino acid sequence of the N‐terminal tryptic glycopeptide from a minor human erythrocyte membrane sialoglycoprotein (component D or glycophorin C) was determined by manual sequencing. The glycosylation sites were identified by a new procedure for the detection of the glycosylated derivatives released by Ednian degradation. The fragment, comprising 47 residues, was found to contain an average of about 12 0‐glycosidically linked oligosaccharides and one asparagine‐linked carbohydrate chain. An identical hexapeptide sequence occurring in two regions of the glycopeptide provides evidence that it has developed by an internal gene duplica‐ tion during evolution. In addition, a part of its structure shows a striking similarity to the sequence of a certain region of the MN and Ss erythrocyte membrane sialoglycoproteins (glycophorins A and B), suggesting that the molecules might be related.
DNA Synthesis at a Fork in the Presence of DNA Helicases
Authors: KUHN Bernhard , ABDEL‐MONEM Mahmoud
Published: March 2005

In a mixture of Escherichia coli DNA polymerase III holoenzyme, single‐strand‐binding protein, artificially forked λ bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicaseII, less so by helicases I, III or rep protein of E. coli or T4 phage helicase. The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA poly‐ merase I or T4 DNA polymerase. In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA. We believe that helicase II both shifts the forks and stimulates DNA polymerase III. The results support the conclusion derived from previous studies that helicase II is part of the DNA‐synthesizing system of E. coli.
Characterization of the Active Site of Ribonucleotide Reductase of Escherichia coli, Bacteriophage T4 and Mammalian Cells by Inhibition Studies with Hydroxyurea Analogues
Authors: KJØLLER LARSEN Ingrid , SJÖOBERG Britt‐Marie , THELANDER Lars
Published: March 2005

Hydroxyurea specifically inhibits the enzyme ribonucleotide reductase by inactivating the essential tyrosine free radical in one of the two non‐identical subunits constituting the enzyme. Studies on the reactivity of hydroxyurea analogues towards the free radical salt, potassium nitrosodisulphonate, and the radical‐containing B2 subunit of Escherichia coli ribonucleotide reductase showed that, to obtain maximal enzyme inhibition, the most important parameter of an analogue was the ability to undergo a one‐electron oxidation. Also, a common geometrical feature for the analogues with high inhibitory potency was the planarity of the molecules.

Results of inhibition studies with analogues containing bulky substituents but having about the same ability to reduce the free radical salt suggested that the tyrosine free radical of protein B2 is located in a pocket about 0.4 nm wide and more than 0.6 nm deep

The ribonucleotide reductases of E. coli, T‐4infected E. coli and mammalian cells all contain the same tyrosine free radical structure. Still the mammalian reductase showed a 75‐fold higher sensitivity to inhibition by 3,4‐dihydroxybenzohydroxamic acid than the E. coli reductase. In contrast, the sensitivity to hydroxyurea was the same for both enzymes. The dihydroxybenzohydroxamic acid and hydroxyurea both reacted immediately (t½ > 5) with the free radical salt. Therefore the difference in sensitivity between the mammalian and bac‐ terial reductase most probably reflects different topologies of their active sites with the site of the mammalian enzyme being more exposed.

The T4‐induced reductase showed a 10‐fold increased sensitivity towards both hydroxyurea and the poly‐ hydroxybenzohydroxamic acids compared to the E. coli enzyme, indicating a greater ability of the T4 tyrosine free radical to undergo a one‐electron reduction. A closer understanding of the different topologies of the active sites of different ribonucleotide reductases could be of great value in the design of target‐directed drugs.
Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria
Authors: SCHLEYER Manfred , SCHMIDT Bernd , NEUPERT Walter
Published: March 2005

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation
Methods for the Detection of Single‐Strand Breaks in DNA under Neutral Conditions and Their Application in a Study on the Mechanism of Repair of N‐Methylated Purines in Mouse Cells
Authors: WINTERSBERGER Erhard
Published: March 2005

Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N‐methylated purines produced by methylating agents such as the mutagen and carcinogen N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases. The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis. As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile. In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction. One of these methods makes use of 95 %, formamide at 40°C in place of alkali to denature DNA with pre‐existing single‐strand breaks, the other measures the capacity of DNA scissions with free 3′‐OH groups to act as primer for Eschericlziu coli DNA polymerase I. Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites. Kinetics of production and repair of single‐strand breaks, produced in 3T6 mouse fibroblasts by incubation with N‐methyl‐ N′‐nitro‐ N‐nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation.
Dolichyl‐Phosphate Phosphatase and Dolichyl‐Diphosphate Phosphatase in Rat‐Liver Microsomes
Authors: BELOCOPITOW Enrique , BOSCOBOINIK Daniel
Published: March 2005

Dolichyl‐phosphate phosphatase and dolichyl‐diphosphate phosphatase activities of a liver‐cell microsomal preparation were solubilized by treatment with Triton X‐100. The I00000 × g supernatant was then passed through a column of Sepharose‐4B – concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6‐phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospho‐ lipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X‐100 for the two enzymes. Dolichyl‐phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl‐diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi.

Dolichyl‐diphosphate phosphatase was more inhibited by divalent cations than dolichyl‐phosphate phos‐ phatase. The apparent Km of dolichyl‐phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl‐phosphate phosphatase, but it produced a stronger inhibition on dolichyl‐diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.
Kinetic Studies on the Interactions of Two Forms of Inorganic Pyrophosphatase of Heart Mitochondria with Physiological Ligands
Authors: VOLK Sergej E. , BAYKOV Alexander A. , DUZHENKO Vladlen S. , AVAEVA Svetlana M.
Published: March 2005

A scheme of interactions of Mg2+ ions and their 1 : 1 complex with PPisub (PPiMg′) with two forms of inorganic pyrophosphatase isolated from beef heart mitochondria has been deduced from the analysis of enzyme kinetics at pH varying from 5.6 to 8.5. The scheme implies the existence of two catalytically important metal‐binding sites on the enzyme. The two enzyme forms differ in maximal velocity and affinity for the metal activator. The pH dependence of kinetic parameters suggests that the active form of the substrate is MgP2O2−7. Ca2+ ions strongly inhibit pyrophosphatase activity and the corresponding Hill coefficient is 1.5. Phosphate and ATP are weak inhibitors of pyrophosphatase of the competitive and noncompetitive type respectively. The results show that these forms of mitochondria1 pyrophosphatase are similar to pyrophosphatases isolated from other sources.
Kinetic and Structural Differences between Cytochrome c Oxidases from Beef Liver and Heart
Authors: MERLE Peter , KADENBACH Bernhard
Published: March 2005

The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eight‐ fold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content.

The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c‐depleted membranes, deoxycholate‐dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low‐affinity and for the high‐affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes.

Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme.

By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase iso‐ enzymes in liver and heart is discussed.