bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐linked proteins associated with a specific mRNA in the cytoplasm of HeLa cells
Authors: RUZDIJIC Sabera , BAG Jnanankur , SELLS Bruce H.
Published: March 2005

Cytoplasmic messenger RNAs of eukaryotic cells are distributed between polysomal and post‐polysomal fractions (free) as protein‐bound complexes. These studies were designed to determine whether a specific mRNA isolated from difrerent subcellular compartments is Complexed with the same family of polypeptides. As a first approach we have examined the proteins associated with mRNA which codes for histone H4. To perform these experiments HeLa cells were exposed to ultraviolet light to cross‐link in vivo polypeptides which are closely associated with nucleic acid. To identify the polypeptides associated with mRNA specific for histones a genomic probe for histone H4 mRNA was immobilized on epoxy‐cellulose. By hybrid selection specific mRNPs containing histone mRNA were isolated. Our results reveal the existence of a number of polypeptides associated with both polysomal and post‐polysomal histone mRNAs. In polysomal histone mRNA two polypeptides of Mr= 49000 and 52500 were the major components. In contrast polypeptides of Mr= 43000 and 57000 were the major polypeptide components of post‐polysomal (or free) histone mRNA. Furthermore, these results also suggest that the polypeptides associated with either polysomal or free H4 histone mRNA represent a subset of proteins found in poly(A)‐free fractions or poly (A)‐rich mRNA fractions.
Analysis of the control of citrulline synthesis in isolated rat‐liver mitochondria
Authors: WANDERS Ronald J. A. , VAN ROERMUND Carlo W. T. , MEIJER Alfred J.
Published: March 2005

The amount of control of the various steps involved in the citrulline synthesizing pathway in isolated rat‐liver mitochondria incubated with saturating concentrations of ammonia and ornithine has been measured.

The flux control coefficient of carbamoyl‐phosphate synthase (ammonia) was close to one. Control exerted by other steps in the pathway, including ornithine transcarbamoylase, carbonic anhydrase, ATP production and transport of ornithine, was negligible.

The high flux control coefficient of carbamoyl‐phosphate synthetase is due to the low elasticity coefficient of this enzyme towards its product, intramitochondrial carbamoyl phosphate, in combination with a high elasticity coefficient of ornithine transcarbamoylase towards carbamoyl phosphate.
Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP‐dependent protein kinases
Authors: DE WIT René J. W. , HEKSTRA Doeke , JASTORFF Bernd , STEC Wojciech J. , BARANIAK Janina , VAN DRIEL Roel , VAN HAASTERT Peter J. M.
Published: March 2005

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP‐binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen‐bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2′, 3′ and 5′ positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation.

The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocydic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)‐adenosine 3′, 5′‐monophosphorothioate [Rp−cAMPS], in total absence of activation even at concentrations, at which the analog saturates both binding sites. Since occupancy of the cAMP‐binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)‐cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)‐cAMPS showed antagonistic activity, while with a cAMP‐dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed.

Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et al. (1982) Eur. J. Biochem. 122, 95‐99]. This was based on measurements with impure preparations of (Rp)‐cAMPS and the Rp and Sp isomers adenosine 3′, 5′‐monophosphodimethylamidate, cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)‐cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.
The distance between S1, S21, and the 3′ end of 16S RNA in 30S ribosomal subunits
Authors: ODOM Obed W. , DABBS Eric R. , DIONNE Craig , MÜLLER Merold , HARDESTY Boyd
Published: March 2005

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3′ end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non‐radiative energy transfer to be: S21–16S RNA, 5.1 nm; S21–S1, 6.9 nm; S1–16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1–S21 distance. Binding of 50S subunits causes an apparent increase in the S21 –16S RNA and S21–S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1–16S‐RNA distance.
Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose :H+ carrier
Authors: SECKLER Robert , WRIGHT J. Keith
Published: March 2005

The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C‐terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I‐labelled anti‐(C‐terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I‐labelled anti‐(C‐terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside‐out vesicles prepared by high‐pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles.

Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right‐side‐out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high‐pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right‐side‐out vesicles.

In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside‐out and right‐side‐out populations while maintaining 41% in the right‐side‐out orientation.

Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside‐out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti‐(C‐terminus) antibodies, which can, in principle, be used in Method I. Modification of Method II permits the determination of carrier levels in vesicles prepared from cells by high‐pressure lysis with as little as 0.1 pmol of carrier to an accuracy of 30–35%.

The functional symmetry of certain aspects of carrier‐catalyzed facilitated diffusion and active transport is discussed in the context of the nonpreferential, bimodal insertion of the carrier into proteoliposomes.
Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics
Authors: OKAJIMA Kenji , INOUE Masayasu , MORINO Yoshimasa , ITOH Kazunobu
Published: March 2005

Acetylation of cysteine S‐conjugates of xenobiotics by microsomal N‐acetyltransferase is the final step of detoxicative metabolism leading to mercapturic acid biosynthesis. To elucidate the subcellular site of N‐acetylation and the effective mechanism by which the final metabolites are eliminated from the organisms, topological aspects and catalytic properties of microsomal N‐acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. Intravenous administration of radioactive S‐benzyl‐L‐cysteine, a model compound of cysteine S‐conjugates, resulted in rapid acetylation of the conjugate in liver and kidney to a similar extent. The acetylation was followed by a rapid excretion of the metabolite, a mercapturic acid, into the urine; about 60% of the injected dose appeared in urine within 60 min of administration.

Limited proteolysis of microsomal vesicles obtained from liver and kidney by chymotrypsin or trypsin inactivated the transferase by 49–62% and 62–73%, respectively. Proteolytic inactivation of the transferase was not significantly affected by the presence of 0.04% sodium deoxycholate by which the vesicles became permeable to macromolecules due to its detergent action.

To determine the sidedness of the active site of N‐acetyltransferase on the microsomal membranes, two S‐acetyldextran polymer derivatives (Mt 500000) of cysteine and N‐acetylcysteine which represent an nonpermeant substrate and product for this enzyme, respectively, were examined for their effects on the vesicle‐associated enzyme activity. Both derivatives inhibited the transferase activity in a dose‐dependent fashion; maximum inhibition of the enzyme activity was 40% by the former and 60% by the latter.

Sulfobromophthalein strongly inhibited the enzyme activity and, this inhibition was completely reversed by adding an equimolar amount of hepatic glutathione S‐transferases (ligandins). In contrast to the strong inhibition by sulfobromophthalein itself, its glutathione S‐conjugate did not inhibit the enzyme activity.

These results indicate that the active site and the protease‐sensitive domain(s) of the microsomal N‐acetyltransferase are localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and that the ligandin(s) might protect membranous N‐acetyltransferase‐from inhibition by organic anions by binding them and catalyzing the conjugation with glutathione.
The use of nucleotide phosphorothioate diastereomers to define the structure of metal‐nucleotide bound to GTP‐AMP and ATP‐AMP phosphotransferases from beef‐heart mitochondria
Authors: TOMASSELLI Alfredo G. , MARQUETANT Rainer , NODA Lafayette H. , GOODY Roger S.
Published: March 2005

The diastereomers of adenosine 5′‐O‐[1‐thio]triphosphate (ATP[αS]) and adenosine 5′‐O‐[2‐thio]triphosphate (ATP[βS]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP‐AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5′‐O‐[thio]triphosphate (GTP[αS]) and guanosine 5′‐O‐[2‐thio]triphosphate (GTP[βS]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP‐AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5′‐O‐[1‐thio]diphosphate (GDP‐[αS]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (Vfor isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP‐AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221–227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109–115]. In all cases, coordination of Mg2+ to the β‐phosphate via the pro‐R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP[βS] or GTP[βS] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP[αS] or GTP[αS], or for GDP[αS] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the α‐phosphate group. The observed stereospecificity for the α‐thiophosphate is consistent with the assumption of an interaction of the pro‐R oxygen of the α‐phosphate group with the enzyme.
Increased stability and antiviral activity of 2′‐O‐phosphoglyceryl derivatives of (2′‐5′)oligo(adenylate)
Authors: BAYARD Bernard , BISBAL Catherine , SILHOL Michele , CNOCKAERT Joël , HUEZ Georges , LEBLEU Bernard
Published: March 2005

Metabolically stable analogues of (2′‐5′)oligo(adenylate), (2′‐5′)(A)n. might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2′‐O‐phosphoglyceryl derivatives of (2′‐5′)(A)n oligomers, (2′‐5′)(A)n‐PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell‐free extracts.

In line with its increased stability, (2′‐5′)(A)n‐PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells.

Since (2′‐5′)(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.
Cytochrome b of cob revertants in yeast
Authors: BURGER Gertraud , HOFNER Eva
Published: March 2005

22 revertants of Saccharomyces cerevisiae with intragenic suppressors (sup3) of cob exon mutations (G. Burger. Mol. Gen. Genet., in the press) were analyzed. They display either a reduced amount of cytochrome b, or a shifted maximum absorption wavelength of total cytochrome b or a reduced growth rate on glycerol.

The relationship of physico‐chemical properties (content, light absorption and midpoint potential of cytochromes bK and bT) and functional properties (electron transport and energy yield) has been examined.

In seven of eight revertants with a shifted maximum absorption wavelength of cytochrome b neither growth rate nor electron transfer activity was affected.

In 13 of 14 revertants, reduced content of cytochrome b corresponds to a reduced electron transport rate through the cytochrome bc1 segment.

A lower enzymatic activity, which is not due to a quantitative but to a qualitative alteration of cytochrome b was found in two revertants.

Two revertants show electron transport rates of wild‐type level concomitant with a reduced growth rate on glycerol, obviously due to a less efficient energy coupling. All revertants were shown to contain a high and a low potential cytochrome b, referred to as bK and bT.

Those cob−/supa mutations which shift the maximum absorption wavelength or diminish the content of cytochrome b affect both b cytochromes in all cases. The results support that electron transport and energy conservation are catalyzed by the unity of cytochrome bK and bT and that both heme centers are bound to an identical apoenzyme.

Comparing electron flow rates of succinate:cytochrome c oxidoreductase and NADH:cytochrome c oxido‐reductase in cob mutants and two revertants provides evidence that ubiquinone does not constitute a homogeneous pool, suggested by the dissimilar interaction of both dehydrogenases with the bc1 segment.
Patterns of histone acetylation in Physarum polycephalum
Authors: WATERBORG Jaap H. , MATTHEWS Harry R.
Published: March 2005

Histone acetylation has previously been correlated with both chromosome replication and transcription. We present evidence that (a) confirms both correlations in the true slime mold, Physarum polycephalum and (b) shows that quite a different pattern of acetate turnover is associated with replication compared with transcription. The pattern asssociated with replication involves turnover of acetate on all four core histones on species containing one or two acetates per molecule. This pattern was resolved from the transcription‐associated pattern by three different procedures: (a) detailed analysis of gels of histones pulse‐labelled with acetate; (b) the pattern of acetylation of histones pulse‐labelled with [3H]lysine; and (c) the pattern of acetylation of soluble histones. The pattern associated with transcription is restricted to histones H3 and H4 and occurs mostly on highly acetylated species. This pattern was resolved by (a) analysis of gels of histones pulse‐labelled with acetate; (b) the pattern of histone acetylation in G2 phase of the cell cycle; and (c) the pattern of histone acetylation in the presence of cycloheximide.
Transport of proteins into chloroplasts
Authors: ROBINSON Colin , ELLIS R. John
Published: March 2005

We have partially purified a soluble protease from Pisum sativwn chloroplasts involved in the processing of precursor polypeptides imported into the organelle. The enzyme processes precursors of both stromal and thylakoid proteins to the mature size, but is inactive against all proteins so far tested other than precursors destined for the chloroplast. The enzyme processes precursors from wheat and barley, and is therefore not species‐specific. It has a relative molecular mass of about 180000 and a pH optimum near 9. The enzyme is inhibited by ethylenediamine tetraacetate and 1,10‐phenanthroline but not by serine‐ or thiol‐protease inhibitors.
Transport of proteins into chloroplasts
Authors: ROBINSON Colin , ELLIS R. John
Published: March 2005

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20000) is processed to the mature size (relative molecular mass 14000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr18000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre‐incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate‐treated precursor is taken up and converted to a number of polypeptides of Mr 18000 and below.
Enzymatic isomerization and epimerization of d‐erythrose 4‐phosphate and its quantitative analysis by gas chromatography/mass spectrometry
Authors: OHASHI Kazufumi , TERADA Tomoyuki , KOHNO Takeyuki , HOSOMI Saburo , MIZOGUCHI Tadashi , UEHARA Kihachiro
Published: March 2005

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of d‐erythrose 4‐phosphate to d‐erythrulose 4‐phosphate and d‐threose 4‐phosphate. The presence of d‐erythrulose 4‐phosphate and d‐threose 4‐phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of d‐erythrulose and d‐threose was confirmed by thin‐layer chromatography, gas‐liquid chromatography and an enzymatic method depending upon d‐erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that d‐threose 4‐phosphate was also converted into d‐erythrulose 4‐phosphate and d‐erythrose 4‐phosphate. At the equilibrium, about 90 % of the tetrose 4‐phosphate existed in the form of d‐erythrulose 4‐phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin‐layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4‐phosphate.
Glycosidic linkages of swede cell walls and their residues recovered from the terminal ileum of the pig
Authors: MILLARD Peter , CHESSON Andrew
Published: March 2005

The glycosidic linkage pattern of unfractionated swede cell walls was determined by methylation analysis. The main linkages in descending order of concentration were: (1→4)‐linked glucose, (1→4)‐linked xylose, terminal galactose, (1→ 4)‐linked mannose, terminal arabinose and (1→2)‐linked rhamnose. The major branch points were to the O‐2 or O‐3 atoms of xylose, O‐4 of rhamnose and O‐6 of glucose. The subsequent degradation of swede cell walls by passage through the small intestine of the pig was studied by quantitative methylation analysis of material recovered from animals cannulated 150 mm before the ileo‐caecal junction. In addition to a substantial loss of uronic acid and phenolic material, the proportions of the different glycosidic linkages recovered from the digesta varied greatly from those of the parent material. Pectic polysaccharides had apparent digestibilities in the range of 0.60–0.75 while those of hemicellulosic components varied between 0.15 and 0.57. Glucose units linked 1→4 (with an. apparent digestibility of 0.24) accounted for the greatest weight loss of neutral sugars from the cell wall, followed by terminal galactose, terminal arabinose and 2‐linked arabinose.
Nucleotide sequence of the pyr D gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase
Authors: LARSEN Jørgen Nedergaard , JENSEN Kaj Frank
Published: March 2005

Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Eschrichia coli carrying the pyr D gene on a multicopy plasmid. The preparation had a specific activity of 120 μmol min−1 mg−1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr= 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors.

The complete sequence of 1357 base pairs of an EcoRI‐EcoRI DNA fragment containing the pyr D gene was established. Dihydroorotate dehydrogenase is encoded by a 336‐triplets open reading frame. The molecular mass (Mr= 36732), the amino acid composition and the N‐terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292–303, i. e. Ile‐Ile‐Gly‐Val‐Gly‐Gly‐Ile‐Asp‐Ser‐Val‐Ile‐Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed.

By the S 1‐nuclease technique an mRNA start was mapped 34 ± 2 nucleotide residues upstream of the beginning of the coding frame of pyr D. The leader region contains no similarity to the attenuators of the pyr B and pyr E genes of E. coli.
Multiplicity in celluslases of Schizophyllum commune
Authors: WILLICK Gordon E. , SELIGY Verner L.
Published: March 2005

The white‐rot fungus, Schizophyllum commune, secretes a member of each of three classes of cellulases: a β‐glucosidase, an exoglucanase, and an endoglucanase. Antibodies were developed to members of each of these three enzyme classes. Secretion of these cellulases is induced when a mycelium is ransferred from a glucose to cellulose medium. The maximum level of cellulase transcripts, as indicted by the ability to direct biosynthesis of these cellulases in the rabbit reticulocyte cell‐free translation system, occurred when the rate of secretion was maximum. This implied that initial regulation, at least, of cellulase biosynthesis occurs at the transcriptional level. There were two distinct mRNA‐directed products for each of the cellulases, with sizes estimated to be, for the β‐glucosidase, 95 700 and 93 800, for the exoglucanase, 59 300 and 58 200, and for the carboxymethylcellulase, 40 600 and 39 400. The secreted cellulases are largely glycosylated, as indicated by their binding to concanavalin A and their incorporation of d‐[3H]mannose. The labelled protein was fractionated on concanavalin‐A–agarose; about 70% of the label was bound. A small amount of each of the cellulases appeared in the unbound fraction; the remainder appeared in fractions eluted with 10 mM methyl glucoside or with 100 mM methyl glucoside plus 500 mM methyl mannoside. Thesé results indicated each of the cellulases had an additional heterogeneity in glycosylation, with the most heavily glycosylated and highest molecular weight from eluting last from the concanavalin‐A–agarose. Although tunicamycin (5 μg/ml) blocked glycosylation, there was still some secretion but at a reduced rate which was more pronounced for the β‐glucosidase than for the carboxymethylcellulase activity. The size of the tunicamycin‐secreted product in each case was, within experimental error, equivalent to that of the mRNA‐directed one.
Insulin‐stimulated protein synthesis in adipocytes
Authors: HANSSON Anders , INGELMAN‐SUNDBERG Magnus
Published: March 2005

The mechanisms of insulin stimulation of protein synthesis in adipocytes are presently unknown. Addition of 10 nM insulin to isolated rat adipocytes caused a 1.5–2.5‐fold increase in the protein synthetic rate and a corresponding increase in nascent chain level, indicating that the effect of insulin on protein synthesis in adipocytes is mediated by a stimulation of ribosomal initiation. The effect on protein synthesis exhibited a lag time of 6–8 min after insulin addition. A similar time dependence was also observed for the insulin‐induced phosphorylation of ribosomal protein S6. This supports the proposal that these two phenomena are causally linked.
Solubilization and purification of the ATPase from the tonoplast of Hevea
Authors: MARIN Bernard , PREISSER Joachim , KOMOR Ewald
Published: March 2005

The tonoplast‐bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100‐fold with two ammonium sulfate precipitation steps and a G‐200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 100 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54‐kDa and the 66‐kDa polypeptides exist in multiple copies. No close resemblance to the membrane‐bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody crossreaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase.

Many properties of the tonoplast‐bound ATPase such as pH‐dependence, substrate specificity, ion‐dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP‐hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4′‐diisothiocyano‐2,2′‐disulfonic acid stilbene, which are strong inhibitors of membrane‐bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.
Amino acid sequence of histone H1 at the ADP‐ribose‐accepting site and ADP‐ribose · histone‐H1 adduct as an inhibitor of cyclic‐AMP‐dependent phosphorylation
Authors: USHIROYAMA Takahisa , TANIGAWA Yoshinori , TSUCHIYA Mikako , MATSUURA Ryoji , UEKI Minoru , SUGIMOTO Osamu , SHIMOYAMA Makoto
Published: March 2005

The ADP‐ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP‐ribosyltransferase was determined and effects of the ADP‐ribose · histone‐H1 adduct on cAMP‐dependent phosphorylation of the histone H1 were investigated.

ADP‐ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate‐32P]NAD and the purified ADP‐ribosyltransferase. N‐Bromosuccinimide‐directed bisection of ADP‐ribosylated histone H1 showed that the NH2‐terminal fragment (Mr= 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP‐dependent protein kinase. Digestion of the NH2‐terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high‐performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29–34 of histone H1, containing the arginine residue as the ADP‐ribosylation site. These results indicate that ADP‐ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2‐terminal side of the phosphate‐accepting serine residue 38.

Phosphorylation of histone H1 from calf thymus by cAMP‐dependent protein kinase was markedly reduced when histone H1 was ADP‐ribosylated. Kinetic studies of phosphorylation revealed that ADP‐ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non‐competitive inhibitor of ATP.
Thrombin specificity
Authors: CHANG Jui‐Yoa
Published: March 2005

α‐Thrombin cleavage of 30 polypeptide hormones and their derivatives were analysed by quantitative aminoterminal analysis. The polypeptides included secretin, vasoactive intestinal polypeptide, cholecystokinin fragment, dynobrphin A, somatostatins, gastrin‐releasing peptide, calcitonins and human parathyroid hormone fragment. Most of them were selected mainly on the ground that they contain sequence structures homologous to the well known tripeptide substrates of α‐thrombin. All selected polypeptides have one single major cleavage site and both Arg‐Xaa and Lys‐Xaa bonds were found to be selectively cleaved by α‐thrombin. Under fixed conditions (1 nmol polypeptide/0.5 NIH unit α‐thrombin in 20 μl of 50 mM ammonium bicarbonate at 25°C), the time required for 50% cleavage ranges from less than 1 min to longer than 24 h. Heparin invariably enhanced thrombin cleavage on all polypeptide analysed. The optimum cleavage site for α‐thrombin has the structures of (a) P4‐P3‐Pro‐Arg‐P1′‐P2′, where P3 and P4 are hydrophobic amino acid and P1′, P2′ are nonacidic amino acids and (b) P2‐Arg‐P1′, where P2 or P1′ are Gly. The requirement for hydrophobic P3 and P4 was further demonstrated by the drastic decrease of thrombin cleavage rates in both gastrin‐releasing peptide and calcitonins after chemical removal of hydrophobic P3 and P4 residues. The requirement for nonacidic P1′ and P2′ residues was demonstrated by the drastic increase of thrombin cleavage rates in both calcitonin and parathyroid hormone fragments, after specific chemical modification of acidic P1′ and P2′ residues. These findings confirm the importance of hydrophobic P2–P4 residues for thrombin specificity and provide new evidence to indicate that apolar P1′ and P2′ residue are also crucial for thrombin specifity. It is concluded that specific cleavage of polypeptides by α‐thrombin can be reasonably predicted and that chemical modification can be a useful tool in enhancing thrombin cleavage.
Thrombin specificity
Authors: CHANG Jui‐Yoa , ALKAN Sefik S. , HILSCHMANN Norbert , BRAUN Dietmar G.
Published: March 2005

Selective cleavage of polypeptides by α‐thrombin can be reasonably predicted [Chang, J.‐Y. (1985) Eur. J. Biochem. 151, 217–224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse k light chains 10K26 and 10K44 from anti‐(azobenzene arsonate) antibodies contain 20 Arg/Lys‐Xaa bonds. Only two of them, one ProArg‐Thr bond located at the joint of the variable region with the joining peptide and one ValLys‐Ser bond located near the carboxyl‐terminal end of the constant region, were selectively cleaved by α‐thrombin. The ProArg‐Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys‐Ser has a 50% cleavage time approx. 9–10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short‐time thrombin digestion. Fragments containing intact variable region (1–96) and intact joining peptide–constant region (97–214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) λ light chains from both human and mouse all begin with the GlnProLys‐(Ala/Ser) structure (positions 108–111) at their constant regions. This ProLys‐Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human λ chain (KERN, NEI, NEW, VOR) and one mouse λ chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys‐Ala/Ser bonds. For human λ chains, the 50% cleavage time at this ProLys‐Ala bond was approx. 3–4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC‐20. (c) Heparin inhibits thrombin cleavage of Arg/Lys‐Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl‐terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by α‐thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human‐mouse light chain chimeras.
Identification and characterization of the principal proteins of the fat‐globule membrane from guinea‐pig milk
Authors: JOHNSON Virginia G. , GREENWALT Dale E. , HEID Hans W. , MATHER Ian H. , MADARA Patricia J.
Published: March 2005

The milk‐fat‐globule membrane (MFGM) was isolated from guinea‐pig milk and the membrane‐associated proteins and glycoproteins characterized by electrophoretic techniques. Major components of the membrane included PAS‐I, a sialoglycoprotein of Mr≥ 200000, the redox enzyme xanthine oxidase and the glycoprotein, butyrophilin. Membrane preparations also contained two other glycoproteins, GP‐80 and GP‐55, of Mr 80000 and 55000, respectively.

Comparison of guinea‐pig xanthine oxidase and butyrophilin with proteins from bovine MFGM by peptide mapping procedures, showed that the two proteins in both species were similar, but not identical. GP‐55 may also be related to glycoproteins of Mr 45000 and 48000 in the bovine membrane.

The integral and peripheral components of guinea‐pig MFGM were identified by treating membrane preparations with sodium carbonate solutions at high pH and by partitioning the membrane proteins in solutions of Triton X‐114. By these criteria xanthine oxidase and GP‐55 appeared to be peripheral components and GP‐80 an integral protein of the membrane. PAS‐I and butyrophilin displayed hydrophilic properties in Triton X‐114 solutions, but could not be removed from membrane preparations with sodium carbonate. Possible reasons for these ambiguous data are discussed.

The observed similarity between several of the proteins of guinea‐pig and bovine MFGM implies that these proteins may have specific functions related to milk secretion in mammary tissue, e.g. in the budding of milk‐fat globules or the exocytosis of milk protein and lactose at the apical surface.
The redox interconversion mechanism of Saccahromyces cerevisiae glutathione reductase
Authors: PINTO M. Carmen , MATA Ana M. , LÓPEZ‐BAREA Juan
Published: March 2005

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s).

The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH‐binding site, although interfering with transhydrogenase activity, perhaps by conformational changes.

The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580–600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge‐transfer complex between the proximal thiolate and oxidized FAD in the two‐electron‐reduced enzyme.

The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half‐cystines located at the GSSG‐binding site and another cysteine nearby.
Poly(l‐proline)‐binding proteins from chick embryos are a profilin and a profilactin
Authors: TANAKA Masato , SHIBATA Hiroko
Published: March 2005

Two poly(l‐proline)‐binding proteins (PBP‐1 and PBP‐2) were purified from chick embryos by using a poly(l‐proline)‐agarose column. PBP‐1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1:1. The other poly(l‐proline)‐binding protein, PBP‐2, was the 15‐kDa protein itself. The 42‐kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self‐polymerize. In addition, the 42 + 15‐kDa protein complex (PBP‐1) inhibited DNase I, just as a monomeric actin did. The amino acid composition of the 15‐kDa protein was similar to that of mammalian profilin and it inhibited the salt‐induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(l‐proline)‐binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(l‐proline) must be due to profilin itself, because the profilin has a greater affinity for poly(l‐proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(l‐proline).
Chemical modification of the bifunctional human serum pseudocholinesterase
Authors: BOOPATHY Rathanam , BALASUBRAMANIAN Aiylam S.
Published: March 2005

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2‐hydroxy‐5‐nitrobenzyl bromide, N‐bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenyglyoxal and 2,4‐pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N‐Ethylmaleimide and p‐hydroxy‐mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase.

The use of N‐acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation.

Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imimpramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2‐hydroxy‐5‐nitrobenzyl bromide, N‐bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N‐acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non‐active‐site tyrosine residues.
An improved procedure for purifying 5′‐nucleotidase from various sources
Authors: DIECKHOFF Josef , KNEBEL Helmut , HEIDEMANN Martin , MANNHERZ Hans Georg
Published: March 2005

5′‐Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5′‐nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E. M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J. P. (1984) Biochem. J. 221, 369–377). Gel filtration, using non‐denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5′‐nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5′‐nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5′‐nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F‐actin than the enzyme isolated from rat liver.
Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer
Authors: SMITH Stuart , STERN Alan , RANDHAWA Zafar I. , KNUDSEN Jens
Published: March 2005

We have explored a comprehensive experimental approach to determine whether the two condensing‐enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95‐kDa polypeptides. These core polypeptides lack the chain‐terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long‐chain acyl moieties at a rate equal to that of the native enzyme. The 4′‐phosphopantetheine content of these enzyme preparations, estimated from the amount of β‐alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2‐14C]acetate, was typically 0.86 mol/mol 95‐kDa polypeptide. The stoichiometry of long‐chain acyl‐enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl‐chains were assembled/mol 95‐kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125‐kDa polypeptide of one subunit could be cross‐linked to the 95‐kDa polypeptide of the other subunit by 1,3‐dibromo‐2‐propanone yielding a single molecular species of 220 kDa. Cross‐linking was accompanied by a loss of condensing‐enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J. K. Stoops and S. J. Wakil (1981) J. Biol. Chem. 256, 5128–5133] in which the juxtaposed 4′‐phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross‐linking. Both half‐maximal cross‐linking and 50% inhibition of activity were observed with 1 mol 1,3‐dibromo‐2‐propanone bound/mol enzyme. After assembly of long‐chain acyl moieties on the 4′‐phosphopantetheine residues, no vacant condensing‐enzyme active sites were demonstrable either by cross‐linking with 1,3‐dibromo‐2‐propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate.

These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.