bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteoglycans of Guinea‐Pig Costal Cartilage
Authors: LOHMANDER Stefan 
Published: March 2005

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea‐pigs. Three solvents were used in sequence: 0.4 M guanidine · HCl, pH 5.8, 4 M guanidine · HCl, pH 5.8, and 4 M guanidine · HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4‐sulfate to chondroitin 6‐sulfate. Amino acid analyses of dissociative fractions were in accordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6‐sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts.The results indicate that the proteoglycans of guinea‐pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.
The Mechanism of the Halothane‐Dependent Efflux of Calcium from Rat‐Liver Mitochondria
Authors: GRIST Elizabeth M. , BAUM Harold 
Published: March 2005

The halothane‐dependent, calcium‐induced loss of respiratory control in rat liver mitochondria [1,2] is Mg2+‐dependent and is accompanied by an enhanced mitochondrial swelling. It is suggested that this swelling reflects an increase in calcium activity in the matrix space, due to a decrease in binding of the accumulated cation. This change in the partition of intramitochondrial calcium is correlated with an inhibition by halothane of energy‐independent, calcium‐induced swelling.The enhanced swelling associated with the active accumulation of calcium in the presence of halothane does not lead to a marked increase in permeability to other ions. Nevertheless, under conditions of energised calcium uptake, and in the presence of Mg2+, a halothane‐dependent, ruthenium red‐insensitive efflux of calcium is observed. This is consistent with the proposed halothane‐dependent increase in the matrix activity of accumulated Ca2+. It is suggested that this mechanism accounts for the previously postulated [2] futile cycle of calcium uptake and release induced by halothane in rat liver mitochondria.
Lysosomal Enzymes as Agents of Turnover of Soluble Cytoplasmic Proteins
Authors: DEAN Roger T. 
Published: March 2005

The degradation of cytosol proteins in vitro by purified cathepsin D and cathepsin B1 and by mixtures of lysosomal enzymes, was studied. By means of a double‐labelling method, it was shown that the relative rates of degradation of cytosol proteins by the purified enzymes and by mixtures of enzymes under a wide range of conditions in vitro correlated well with their relative rates of turnover in vivo.The complex mixture of cytosol proteins was degraded less rapidly after denaturation than in the native state, both by the purified proteases and by the mixture of lysosomal enzymes. This contrasts with previous results on proteolysis of single purified proteins. The possible role of lysosomal enzymes in turnover in vivo was discussed.
Studies on Plasma Free‐Fatty‐Acid Metabolism and Triglyceride Synthesis of Brown Adipose Tissue in vivo during Cold‐Induced Thermogenesis of the Newborn Rabbit
Authors: SCHENK Herwart , MENDE Thomas , GOETZE Eberhard , HEIM Tibor , VARGA Ferenc 
Published: March 2005

Parameters of plasma free fatty acid metabolism (pool size, half time, disappearance rate, turnover time and absolute turnover rate), the influx of plasma free fatty acids into the glycerides of brown adipose tissue and the pathway of triglyceride synthesis in brown adipose tissue (glycerol‐1‐phosphate versus monoglyceride pathway) were examined after intravenous injection of [1‐14C]palmitate in newborn rabbits. In the thermoneutral environment of 35°C the turnover rate of plasma free fatty acids was 10.20 μmol/min per 100 g body weight and its flux into the glycerides of brown adipose tissue 0.367 μmol/min per 100 g body weight. Cold exposure at an ambient temperature of 20°C caused a decrease to 5.84 μmol/min and 0.207 μmol/min per 100 g body weight, respectively.Both under basal conditions at an ambient temperature of 35°C and under cold‐induced thermogenesis at an ambient temperature of 20°C triglyceride synthesis in brown adipose tissue ran through the glycerol 1‐phosphate pathway.
Biochemical Research on Oogenesis
Authors: DENIS Herman , MAZABRAUD André , WEGNEZ Maurice 
Published: March 2005

The properties of tRNA from oocytes of Xenopus laevis were compared with those of tRNA from somatic cells of the same species. Both types of tRNAs were found to have the same average length and to contain an equal variety of modified nucleotides. However, tRNA from small oocytes differed from somatic tRNA by its chromatographic behavior on methylated‐albumin kieselguhr columns.The elution profiles from reversed‐phase chromatography 5 columns of several aminoacyl‐tRNAs were compared after charging somatic and oocyte tRNAs with 3H or 14C‐labelled amino acids. Striking differences in peak position were observed when tRNA from small oocytes was mixed and co‐chromatographed with somatic tRNA. The differences were less important when tRNAs from large oocytes and from somatic cells were compared. Mixtures of egg and somatic tRNAs gave completely or almost completely coincident elution profiles. Only one isoacceptor, tRNA1Met (initiator tRNA) had the same position in all reversed‐phase chromatography 5 chromatograms.The results are discussed in terms of possible post‐transcriptional modifications of tRNA in the course of oogenesis. An alternative explanation resorting to changes in the tRNA population of the growing oocyte is also envisaged.
Characterization of Yeast Ribosomal DNA
Authors: RETÈL Jan , VAN KEULEN Harry 
Published: March 2005

High‐molecular‐weight yeast ribosomal DNA, purified by preparative Hg2/Cs2SO4 gradient centrifugation, was characterized using RNA‐DNA hybridization, CsCl gradient analysis and denaturation‐renaturation experiments. The following conclusions are drawn from the results of these studies.

55–60% of the total yeast rDNA is made up of sequences coding for 42‐S ribosomal precursor RNA.
The mean length of the 42‐S precursor cistrons corresponds to a molecular weight of 6.2 × 106.
The overall G + C content of yeast rDNA is 42%. On the other hand, both the segments coding for the nonribosomal excess RNA of the 42‐S precursor and those specifying 26‐S and 17‐S rRNA have a higher G + C content of 46.9–47.5%.
The 42‐S precursor cistrons are closely bound up with sequences rather low in G + C content (34–35%), which account for 40–45% of the total yeast rDNA. In contrast to the multiple 42‐S precursor cistrons which show very little or no base sequence divergence these intervening (A + T)‐rich sequences display a considerable heterogeneity in base sequence.
The Location and Function of Cytochrome c2 in Rhodopseudomonas capsulata Membranes
Authors: HOCHMAN A. , FRIDBERG I. , CARMELI C. 
Published: March 2005

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate.Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9‐aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift.It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophores and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.
Carboxymethylation of Horse‐Liver Alcohol Dehydrogenase in the Crystalline State
Authors: ZEPPEZAUER Eila , OHLSSON Ingrid , JÖRNVALL Hans 
Published: March 2005

Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys‐46, which is a ligand to the active‐site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C‐labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP‐ribose, Pt(CN)2‐4, 1,10‐phenanthroline, Au(CN)‐2 and AMP also prevented alkylation with decreasing effectiveness.Crystallographic studies of the alkylated enzyme show that the carboxymethylated sulfur atom of Cys‐46 is still liganded to the active‐site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN)2‐4 to the enzyme. It was found that Arg‐47 interacts with the phosphate moiety of the nucleotide. Lys‐228 and Arg‐47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano‐ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active‐site zinc atom.The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion‐binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP‐ribose, Pt(CN)2‐4 and to a lesser extent Au(CN)‐2 and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10‐Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc.Identification of this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active‐site zinc region and the general anion‐binding site are also presented.
Modulation of the Enzymic Activity of Chicken Pepsin by the Covalent Modification of Its Single ‐ SH Group
Authors: SHECHTER Yoram , RUBINSTEIN Menachem , BECKER Rosette , BOHAK Zvi 
Published: March 2005

The single cysteinyl residue of chicken pepsin was modified with a wide spectrum of reagents to produce mixed disulfides or alkylated derivatives. All these derivatives showed enhanced catalytic activity towards the synthetic peptide Z‐Ala‐Ala‐Phe‐Phe‐OPrP, where OPrP is the 3‐(4‐pyridyl)‐propyl‐1‐oxy group. The overall catalytic constant kcat/Km for these derivatives was 4‐25‐fold larger than that of the native enzyme. The activity of the enzyme towards denatured hemoglobin was slightly decreased (10–45%) by these modifications. When the mixed disulfide derivatives were treated with excess mercaptan, the sulfhydryl group was regenerated and activity reverted to that of the native enzyme.The ‐SH group of chicken pepsin reacted preferentially with reagents containing an aromatic group. The reaction was found to depend on the ionization of a single group, presumably the ‐SH itself, with a pKa= 7.5. The rate of reaction of the fully deprotonated species with various disulfides was 100–1000‐fold smaller than that of the ‐ SH group of glutathione.It is suggested that the groups attached covalently to the sulfhydryl also interact with other amino acid side chains in the protein thereby affecting the active center of chicken pepsin.
Ion‐Binding to Phospholipids
Authors: HAUSER Helmut , PHILLIPS Michael C. , LEVINE Barry A. , WILLIAMS Robert J. P. 
Published: March 2005

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations > 0.1 mM. 1H and 31P high‐resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid.The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1–10.Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+‐lecithin interactions.An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA∼ 30 l/mol was calculated at lanthanide concentrations > 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding.The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cationlecithin interaction.From spin‐lattice relaxation‐time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site.The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site.The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions.Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.
Identity and Activities of Lysosomal Enzymes in Parenchymal and Non‐Parenchymal Cells from Rat Liver
Authors: VAN BERKEL Theodorus J. C. , KRUIJT Johan K. , KOSTER Johan F. 
Published: March 2005

Intact parenchymal and non‐parenchymal cells were isolated from rat liver. The parenchymal cells were purified by differential centrifugation, while non‐parenchymal cells were obtained free of parenchymal cell contamination by preferentially destroying the parenchymal cells with the aid of pronase (0.25%).
The ability to isolate pure intact parenchymal and non‐parenchymal cells permitted the characterization and measurement of specific activities of various lysosomal enzymes, representing the main functional hydrolytic activities of the lysosomes in these distinct cell types.
Lysosomal enzymes catalysing the hydrolysis of the terminal carbohydrate moiety of glycoproteins and glycolipids were not particularly enriched in the non‐parenchymal cells as compared to parenchymal cells. The ratio of the specific activities of non‐parenchymal cells over parenchymal cells varied between 0.7 for N‐acetyl‐β‐d‐hexoseaminidase to 2.1 for α‐glucosidase. This suggests no specific role of the non‐parenchymal cells in the hydrolysis of terminal carbohydrate moieties of glycoproteins and glycolipids.
The enzymes acid phosphatase and aryl sulphatase, representing the phosphate and sulphate hydrolyzing activities, were enriched in the non‐parenchymal cells as compared to the parenchymal cells by a factor of 2.5.
The most important peptidase cathepsin D, representing protein breakdown capacity, is enriched in the non‐parenchymal cells as compared to parenchymal cells by a factor 6.0, suggesting a possible specific function of non‐parenchymal cells in protein breakdown.
The most enriched lysosomal enzyme, representing lipid hydrolysis, is acid lipase, which is enriched in the non‐parenchymal cells with a factor of 10.
The distribution of lysosomal enzymes between parenchymal and non‐parenchymal cells suggests different functional roles of the lysosomes in these cell types. It can be concluded that the non‐parenchymal cells possess a set of lysosomal enzymes which makes them extremely suitable for a phagocytic and antimicrobial function in the liver.
Control of Fatty‐Acid‐Synthetase Biosynthesis in Saccharomyces cerevisiae
Authors: DIETLEIN Gerhard , SCHWEIZER Eckhart 
Published: March 2005

143 out of 308 fasl mutants (47%) and 139 out of the 443 fas2‐mutants (32%) genetically studied in this laboratory fail to complement with any other fas‐mutant (deficient in fatty acid synthetase) of the same gene locus. From these noncomplementing fas‐mutants no mutant fatty acid synthetase can be isolated using the wild‐type enzyme purification procedure. Furthermore the noncomplementing fas‐mutants generally contain no material immunologically crossreacting with a specific fatty acid synthetase antiserum. However, subunits obtained after dissociation of the complex with sodium dodecylsulfate still cross react with this antiserum. Therefore, it is concluded that noncomplementing fas‐mutants contain no fatty acid synthetase component proteins, though one of the two fas‐loci is mutationally unaffected. This conclusion was further confirmed by 14C‐labeled amino acid incorporation studies which indicated that in noncomplementing fas‐mutants, other than in wild type and complementing fas‐mutant cells, no label was incorporated into fatty acid synthetase subunits or precursor proteins. At nonpermissive temperature, the same biochemical and immunological characteristics were observed with temperature‐sensitive non‐complementing fas‐mutants.These results suggest that noncomplementing fas‐mutants either represent regulatory mutants unable to induce the mutationally unaffected other fas‐gene locus or that they are association‐defective mutants. In both cases the resulting individual subunits of the complex may be rapidly degraded by intracellular proteases.
Immunochemical Studies of Factor V
Authors: GREENQUIST Alfred C. , WEINBERG Robert M. , KUO Alice L. , COLMAN Robert W. 
Published: March 2005

Native bovine factor V exhibits a molecular weight of 300000 as determined by gel filtration of untreated plasma. Highly purified factor V exhibits multiple molecular weight forms which range from small active fragments to aggregates of several million which are generated during the purification on cellulose phosphate. Isoelectric focusing of a single high‐molecular‐weight species produced a single protein and activity peak at pH 4.65. Factor V activity is associated with each protein band observed following polyacrylamide gel electrophoresis. Antisera to factor V prepared in rabbits produces a time‐dependent and concentration‐dependent inhibition of factor V activity in plasma and purified factor V. The multiple molecular weight forms of factor V appear equivalent upon immunodiffusion and on immunoelectrophoresis migrate as an alpha globulin between albumin and fibrinogen. Immunoprecipitation arcs are equivalent in plasma and serum.Factor V consists of two major types of subunits, a light chain (73000), aggregates of which form the higher‐molecular‐weight species, and a heavy chain (125000). Using preparations containing one or both chains isolated by disc gel electrophoresis, antiserum was shown to contain two families of antibodies, one against each subunit. Cross reactivity with both light and heavy chain antigens is observed in sheep and goat but not monkey or human plasma. The antisera also neutralized goat and sheep factor V activity.
Animal DNA‐Dependent RNA Polymerases
Authors: HOSSENLOPP Paul , CHAMBON Pierre , WELLS David 
Published: March 2005

DNA‐dependent RNA polymerase was solubilized from normal and adenovirus‐2 infected HeLa cells. Multiple peaks of enzyme activity were separated by DEAE‐Sephadex chromatography. In addition to class A and B enzyme activities (respectively insensitive and sensitive to inhibition by 10 nM α‐amanitin), three peaks of class C enzyme activity were found which are sensitive to inhibition by α‐amanitin only at much higher concentrations (0.1 mM). Rechromatography of these class C peaks indicates that they are not chromatographic artifacts. Class C enzymes differ from class A and B enzymes by several criteria including inhibition by α‐amanitin, immunological properties, and the ability to transcribe native calf thymus DNA at high ionic strength. However, the ionic strength optimum and the divalent cation requirements of class C enzymes are not invariant characteristics of the enzymes and are markedly dependent on the nature and the amount of template in the reaction. No differences, either qualitative or quantitative, were found between the multiple enzymes isolated from normal or adenovirus‐2 infected cells.All of the partially purified HeLa cell RNA polymerases were able to transcribe an intact double‐stranded adenovirus‐2 DNA under conditions where no transcription occurred with purified calf thymus AI and B RNA polymerases. Since the multiple enzymes were devoid of endonuclease and exonuclease activities, the ability of the partially purified enzymes to transcribe adenovirus‐2 DNA cannot be ascribed to initiation of RNA synthesis at nicks or single‐stranded regions of the DNA. No differences in transcriptional ability were found between corresponding enzyme classes from normal or infected cells, but a comparison of the ability of the various enzyme classes to transcribe intact viral DNA revealed large differences. Although partially purified HeLa class A and B enzymes were able to initiate on the intact viral DNA to a limited extent only, it appears that the class C enzymes transcribe intact duplex DNA much more efficiently than any other class of eukaryotic polymerase yet reported.