bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfer of Proteins across Membranes
Authors: SCHEELE George , DOBBERSTEIN Bernhard , BLOBEL Günter 
Published: March 2005

Canine pancreas was fractionated into free ribosomes and rough microsomes. Detached ribosomes were prepared by treatment of rough microsomes with detergent. Poly(A)‐containing mRNA was extracted from rough microsomes. The biosynthesis of canine pancreatic secretory proteins was studied by comparing proteins synthesized in vitro by translation of mRNA or by completion of nascent chains present in free ribosomes, rough microsomes, and detached ribosomes with proteins synthesized in tissue slices using polyacrylamide gel electrophoresis in sodium dodecyl sulfate and subsequent autoradiography.The banding pattern of authentic secreted proteins synthesized in tissue slices was largely congruent with that obtained from the translation products of rough microsomes indicating that the bulk of the mRNA engaged with rough microsomes codes for secretory proteins.The banding pattern of translation products from mRNA in the absence of microsomal membranes was not congruent with that of authentic secretory proteins. Primary translation products for trypsinogen and the other serine protease zymogens using mRNA appeared to be larger in molecular weight than authentic proteins by 1000–2000 and are thus designated ‘presecretory’ proteins.The banding pattern from the translation products of free ribosomes, which are essentially devoid of membranes, was similar to that of ‘presecretory’ proteins.Translation of mRNA in the presence of microsomal membranes yielded a banding pattern for serine protease zymogens congruent with that of the translation products of rough microsomes, and these products were resistant to posttranslational proteolysis, indicating that segregation and processing of these polypeptide chains had taken place during translation in vitro.
Kinetics of Synthesis of Cytoplasmic Messenger‐Like RNA not Associated with Ribosomes in HeLa Cells
Authors: MAURON Alex , SPOHR Georges 
Published: March 2005

The turn‐over of cytoplasmic messenger‐like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin‐D‐chase experiments, cytoplasmic messenger‐like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients.The half‐life of free mlRNA was found to be of 1–2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short‐lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non‐polyadenylated fractions showed that this short‐lived minor component of half‐life less than 1 h is non‐polyadenylated.
Purification, Biochemical and Immunological Characterisation of Hexosaminidase A from Variant AB of Infantile GM2 Gangliosidosis
Authors: CONZELMANN Ernst , SANDHOFF Konrad , NEHRKORN Heidi , GEIGER Benjamin , ARNON Ruth 
Published: March 2005

Variant AB of infantile GM2 gangliosidosis is a fatal disease leading invariably to death within the first few years of life, due to the excessive storage of the glycolipids GM2 and GA2 which occurs in the nervous tissue of the patient. Unlike other variants of this hereditary disease, where a deficiency of hexosaminidase A, the ganglioside‐GM2‐degrading enzyme, could be demonstrated, the variant AB is characterized by a normal or even elevated level of this enzyme.To examine the possibility of a mutant hexosaminidase A, well capable of hydrolyzing the fluorogenic synthetic substrates but unable to attack the ganglioside, the enzyme was isolated from a patient's tissue and characterized biochemically and immunologically in comparison with an enzyme preparation from normal control tissue. No differences between hexosaminidase A from normal and variant AB tissue could be detected indicating that the defect involved in this disease is not at the genetic level of production of either α or β chains of hexosaminidase A.
The Action of Lysozyme on Partially Deacetylated Chitin
Authors: AMANO Ken‐ichi , ITO Eiji 
Published: March 2005

The effect of N‐unsubstitution of chitin and chitin oligosaccharides on their hydrolysis by lysozyme was studied.
Lysozyme digests of chitin deacetylated at about 70% of its glucosamine residues were fractionated by gel chromatography, paper chromatography and paper electrophoresis, giving six oligosaccharides with N‐unsubstituted glucosamine residues, C1–C6, together with the monomer to tetramer of N‐acetylglucosamine. Oligosaccharides C1, C2 and C3, which accounted individually for about 4–7% of the total glucosamine residues in the isolated saccharides, were identified as GlcNAc‐GlcNAc‐GlcNAc‐GlcN, GlcNAc‐GlcN‐GlcNAc‐GlcNAc and GlcNAc‐GlcN‐GlcNAc‐GlcN, respectively. Oligosaccharide C4, present in a smaller amount, and oligosaccharide C5, present in a very small amount, were identified as GlcNAc‐GlcNAc‐GlcN and GlcN‐GlcNAc‐GlcNAc, respectively. Oligosaccharide C6, accounting for about 7% of the glucosamine residues recovered, was identified as GlcNAc‐GlcN.
On further digestion with excess lysozyme, tetrasaccharide C1 yielded small amounts of N‐acetylglucosamine and GlcNAc‐GlcNAc‐GlcN in addition to major products, (GlcNAc)2 and GlcNAc‐GlcN. Tetrasaccharide C2 was hydrolyzed by lysozyme into GlcNAc‐GlcN and (GlcNAc)2; tetrasaccharide C3 into GlcNAc‐GlcN alone; trisaccharide C4 into N‐acetylglucosamine and GlcNAc‐GlcN. In contrast, trisaccharide C5 was shown to be completely resistant to lysozyme.
The results of digestion of the isolated oligosaccharides with lysozyme, together with the structural feature of these saccharides, were accounted for by the importance of the N‐acetyl groups of the N‐acetylglucosamine residues located on subsites C and E in the enzyme · substrate complexes.
Glycosylation in vitro of Semliki‐Forest‐Virus and Influenza‐Virus Glycoproteins and Its Suppression by Nucleotide‐2‐deoxy‐hexose
Authors: SCHWARZ Ralph T. , SCHMIDT Michael F. G. , LEHLE Ludwig 
Published: March 2005

Cell‐free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]mannose from GDP‐[14C]mannose into dolichol‐phosphate‐mannose, lipid‐linked oligosaccharides and into endogenous virus‐specific glycoproteins.When GDP‐2‐deoxy‐d‐[14C]glucose serves as substrate 2‐deoxy‐d‐[14C]glucose is transferred to dolichol phosphate yielding dolichol‐monophosphate‐2‐deoxy‐d‐[14C]glucose. UDP‐2‐deoxy‐d‐[14C]‐glucose gives rise also to a lipid which, however, is not a polyprenol derivative.The transfer of [14C]mannose to lipid‐extractable fractions and glycoproteins in vitro is blocked by GDP‐2‐deoxy‐d‐glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol‐monophosphate‐[14C]mannose. GDP‐2‐deoxy‐d‐glucose, however, does not inhibit the transfer of [14C]mannose‐labelled oligosaccharides into glycoproteins. UDP‐2‐deoxy‐d‐glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP‐[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2‐deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid‐oligosaccharide.
Active‐Site Catalytic Efficiency of Acetylcholinesterase Molecular Forms in Electrophorus, Torpedo, Rat and Chicken
Authors: VIGNY Marc , BON Suzanne , MASSOULIÉ Jean , LETERRIER François 
Published: March 2005

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O‐ethyl‐S2‐diisopropylaminoethyl methyl‐phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholin‐esterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules.Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 × 107 mol of acetylthiocholine hydrolysed · h−1 at 28 °C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 × 107 mol · h−1), and again lower for rat (1.32 × 107 mol · h−1).In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X‐100) and membrane‐bound enzyme were identical.We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico‐chemical state and physiological properties of its molecular forms.
Affinity Chromatography of Phenylalanine Hydroxylase
Authors: COTTON Richard G. H. , JENNINGS Ian G. 
Published: March 2005

Four independent methods have established that the structure of a previously reported pteridine affinity adsorbent, 6,7‐dimethyl‐5,6,7,8‐tetrahydropterin–CH‐Sepharose, is 5(CH‐Sepharosyl)‐6,7‐dimethyl‐5,6,7,8‐tetrahydropterin.
A novel reaction, the carbodiimide‐promoted coupling of a carboxyl group to N‐5 of a tetrahydropterin, is described.
Two novel adsorbents, 5‐formyl‐tetrahydrofolate–AH‐Sepharose and 5‐methyl‐tetrahydrofolate–AH‐Sepharose, are described which may be useful not only in the study of phenylalanine hydroxylase but also in the study of folate‐metabolizing enzymes.
Carbamoylphosphate Synthetase I of Rat‐Liver Mitochondria
Authors: LUSTY Carol J. 
Published: March 2005

An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two type of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyaacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158000 ± 5000. The weight‐average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8–7.6. Mg2+ and Mn2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn2+ is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP2− concentration is held constant at 1 mM and 10 mM, plots of v versus free Mg2+ concentration are hyperbolic, and extrapolate to zero. These results suggests that free divalent metal ion is required in the forward reaction. The apparent Km of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1–1.5 mM.The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.
Alteration of the Intracellular Concentration of Aminoacyl‐tRNA Synthetases and Isoaccepting tRNAs during Amino‐Acid‐Limited Growth in Escherichia coli
Authors: THOMALE Jürgen , NASS Gisela 
Published: March 2005

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a derepression of the threonyl‐tRNA synthetase and isoleucyl‐tRNA synthetase respectively. During this amino‐acid‐limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed‐phase‐5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl‐tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs.So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibotis. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation signal a meaningful biological function of those newly or increasingly occuring isoaccepting tRNAs.During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine‐limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.
Different Sensitivities of Chloroplasts to Uncouplers when ATP Formation is Induced by Continuous Illumination, by Brief Illumination, by Pre‐Illumination, or by Acid‐Base Transitions
Authors: ORT Donald R. 
Published: March 2005

All of the uncouplers tested are more effective in decreasing ATP formation at the beginning of the illumination period than they are during continuous illumination. The difference is quite small with the uncouplers methylamine and atebrin but very large with the uncouplers octylamine and carbonylcyanide p‐fluoromethoxyphenylhydrazone (FCCP).
The illumination time required to initiate photophosphorylation in the absence of permeant ions is less than 5 ms at high light intensities and the short delay is not much affected by methylamine and atebrin. The delay on the onset of ATP formation is increased to about 40 ms by permeant ions such as K+ in the presence of valinomycin, although the same permeant ions have little or no effect on steady‐state phosphorylation [Ort and Dilley (1976) Biochim. Biophys. Acta, 449, 95–107]. Octylamine and FCCP also delay the onset of phosphorylation for about 40 ms and they do so at concentrations too low to cause significant inhibition of steady‐state phosphorylation. This may be the reason that they are much more inhibitory to phosphorylation during short illumination periods than during longer illumination periods.
The delays in phosphorylation introduced by permeant ions and by low concentrations of the uncouplers octylamine and FCCP are not additive.
Higher concentrations of all of the uncouplers which are capable of causing major inhibitions of steady‐state phosphorylation increase the delay in the onset of phosphorylation, whether or not permeant ions are present. The increase in delay caused by these higher concentrations is a function of the degree of uncoupling and of the extent of the delay already present at low uncoupler concentrations.
For reasons which remain obscure, post‐illumination ATP formation is much more sensitive to the uncoupler methylamine than is ATP formation induced by either continuous illumination or acid‐base transitions.
It is concluded that for the first 40 ms of illumination a transmembrane electric potential gradient is required for photophosphorylation and that permeant ions prevent the formation of such a potential by carrying charges across the lamellar membrane. Presumably the protonated form of octylamine, the octylammonium ion, is itself a permeant cation and therefore functions like K+ in the presence of valinomycin, whereas methylamine once protonated is probably trapped inside the lamellar vesicle, thus preserving the membrane potential. On the other hand, FCCP may destroy any developing membrane potential by carrying outside a small proportion of the hydrogen ions formed inside during electron transport.
During the initial 40 ms of illumination, phosphorylation depends on the chemical potential of the hydrogen ions even though a transmembrane electric potential gradient seems also to be required.
Biosynthesis of the Vacuolar Yeast Glycoprotein Carboxypeptidase Y
Authors: HASILIK Andrej , TANNER Widmar 
Published: March 2005

The vacuolar glycoprotein carboxypeptidase Y (Mr 61 000) is synthesized via a larger precursor (Mr 67 000).
About 35% of the precusor protein can be recovered in the sedimentingfraction of the cell homogenate. A large part of the radioactivity precipitable by antibodies directed against carboxypeptidase Y is redistributed from the sedimenting fraction into the soluble one as the precursor is converted to the enzyme.
The precursor proteinis not identical with the enzyme‐inhibitor complex described previously by Hayashi et al. [Agr. Biol. Chem. 33, 196 (1969)].
The incorporation of radioactivemannose into the precursor indicates that the glycosylation takes place before or immediately after the completion of the syntheses of the polypeptide chain.
The precursor is converted by trypsin in vitro to a protein identical in size to carboxypeptidase Y. The extra piece released by trypsin probably contains four phenylalanine and at least 10 leucine residues.
Of three yeast proteinases tested in vitro only the proteinase B catalyzed conversion of the precursor to a protein identical in size to carboxypeptidase Y.
The conversion in vivo, which presumably is an example of intracellular‐limitedproteolysis. proceeds with a half‐life of about 6 min.
It is assumed that the precursor is an inactive pro‐enzyme, procarboxypeptidase Y, rather than a pre‐enzyme and that, as inactive hydrolase, it exists only during the time required for the intracellular transport to the vacuole, where the pro‐enzyme is activated.
Porcine Follitropin
Authors: CLOSSET Jean , HENNEN Georges 
Published: March 2005

The properties of porcine follitropin and its subunits which have not yet been characterized are presented.The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P‐1 preparation.Its contamination by lutropin and thyrotropin amounted to 1 and 0.5 percent by weight respectively, as measured by radioimmunoassay.The α and β subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography.The amino acid composition of porcine follitropin α subunit was found to be identical to that of α chain of porcine lutropin and thyrotropin.These porcine α chains differ, nevertheless, markedly in their carbohydrate composition particularly with respect to their mannose and galactose contents.The amino‐terminal residue of the follitropin α subunit is threonyl. The carboxy‐terminal end of the α chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin β chain with glutamic acid at its carboxy‐terminal end.Cross‐contamination of the α and β subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively.
The Metabolism of 1‐Phenylethanol and Acetophenone by Nocardia T5 and an Arthrobacter Species
Authors: CRIPPS Roger E. , TRUDGILL Peter W. , WHATELEY John G. 
Published: March 2005

The Arthrobacter sp. is able to grow with (+)‐, (−)‐1‐phenylethanol or the racemic mixture as sole source of carbon. Growth is most rapid with the (−)‐isomer, doubling time 12 h. Enzymes of the already established pathway for acetophenone oxidation are also induced by growth with 1‐phenylethanol and in addition an induced 1‐phenylethanol dehydrogenase that requires NAD as electron acceptor is formed.The 1‐phenylethanol dehydrogenase is active with both 1‐phenylethanol isomers but shows a marked preference for the (−)‐isomer. Relative activity towards the two isomers is mirrored in the rates of growth with the two isomers and the oxidative capabilities of induced cells.The metabolism of the isomers of 1‐phenylethanol by the Arthrobacter sp. is integrated into the established pathway for acetophenone oxidation.The pathway of acetophenone metabolism by Nocardia T5 shows no significant differences from that already established for metabolism of the compound by the Arthrobacter sp. The same spectrum of induced enzymes is formed and the evidence suggests that catechol oxidation again proceeds by ‘ortho’ fission and the β‐oxoadipate pathway, the formation of even transient amounts of cis, cis‐hydroxy muconic semialdehyde has never been observed.In contrast the metabolism of the isomers of 1‐phenylethanol by Nocardia T5 is achieved by a quite different pathway. The side chain is retained without modification during hydroxylation to yield 3‐(1′‐hydroxyethyl)catechol. Ring cleavage, by ‘meta fission’ yields 2,7‐dihydroxy‐6‐oxoocta‐2,4‐dienoate the further fragmentation of which yields lactate and equimolar amounts of pyruvate and acetaldehyde.Growth rates are identical, irrespective of which isomer of 1‐phenylethanol is used as carbon source and the enzymes that convert 1‐phenylethanol to vinyl pyruvate and lactate appear to display no specificity for that chiral centre.
l‐2‐Hydroxytetradecanoic Acid as a Constituent of Salmonella Lipopolysaccharides (Lipid A)
Authors: BRYN Klaus , RIETSCHEL Ernst Th. 
Published: March 2005

l‐2‐Hydroxytetradecanoic acid was recognized as a characteristic, although minor, constituent of the lipid A component of Salmonella lipopolysaccharides. The 2‐hydroxy fatty acid was present in lipid A as an ester, probably bound to the hydroxyl group of some d‐3‐hydroxytetradecanoic acid residues.A survey of enterobacterial lipopolysaccharides showed that l‐2‐hydroxytetradecanoid acid was also present in Klebsiella and Serratia strains. It was absent, however, from lipopolysaccharides of other genera of the family including Escherichia, Shigella, Proteus, Enterobacter and Yersinia. This restricted distribution of the 2‐hydroxy acid may be of significance for taxonomic studies of bacterial genera.