bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal Responsiveness of 3‐Hydroxy‐3‐methylglutaryl‐Coenzyme‐A Reductase and Acetyl‐Coenzyme‐A Carboxylase in Neoplastic Guinea Pig Lymphocytes (L2C)
Authors: PHILIPPOT Jean R. , WALLACH Donald F. H.
Published: March 2005

Hydroxymethylglutaryl‐CoA reductase and acetyl‐CoA carboxylase, rate‐limiting enzymes of cholesterol and fatty acid biosynthesis, respectively, do not show equivalent thermal responsiveness in leukemic L2C guinea‐pig lymphocytes. Both enzymes exhibit Arrhenius plots characteristic of membrane‐association but hydroxymethylglutaryl‐CoA reductase yields a single slope, compared with three slopes, crossing at 24°C and 12°C for acetyl‐CoA carboxylase. Preincubation with phosphatidylcholine liposomes before enzyme assay does not modify the thermal behavior of acetyl‐CoA carboxylase but introduces a discontinuity at 24 C in the Arrhenius diagramin of hydroxymethylglutaryl‐CoA reductase, conferring to this enzyme an activation energy close to that of normal cells.

The Arrhenius plot for fatty acid biosynthesis in L2C cells parallels the thermotropism of the acetyl‐CoA carboxylase, with two discontinuities at 25 and 15 °C. However, cholesterol biosynthesis shows a discontinuity at 24.6 °C, whereas hydroxymethylglutaryl‐CoA reductase activity does not.

The different thermal behavior of hydroxymethylglutaryl‐CoA reductase and acetyl‐CoA carboxylase is discussed in terms of lipid heterogeneities in membrane enzyme environments, i.e. enzyme localization in dissimilar lipid domains. We propose several hypotheses to account for the lack of correlation between the thermotropic responses of hydroxymethylglutaryl‐CoA reductase activity and of cholesterol biosynthesis in L2C cells.
Biochemical Properties and Localization of the Chromosomal Protein IP25
Authors: KEPPEL France , ALLET Bernard , EISEN Harvey
Published: March 2005

The protein IP25, which has previously been reported to accumulate in the chromatin during erythroid differentiation of Friend‐virus‐transformed erythroleukemia cells (FL cells), is shown to behave like histone H1 without being structurally related to it. Like H1. IP25 is not released by digestion of FL cells nuclei with DNAse I. After micrococcal digestion IP25 and HI are differentially distributed in the nucleosome monomers and dimers. This distribution suggests an internucleosomal location for IP25 and H1. Different rates of digestion are observed between nuclei of differentiating and non‐differentiating FL cells with both DNAse I and micrococcal nuclease. These differences could be due to the presence of IP25 in the chromatin of differentiating cells.
Artifacts Imitating Aging of Glucose‐6‐Phosphate Dehydrogenase in Human Erythrocytes
Authors: RȦCZ Oliver , BISZKU Etelka , STRAUB F. Brunó
Published: March 2005

Data in the literature based on the technique of graded osmotic hemolysis have been re‐evaluated. Differences were previously found in the glucose‐6‐phosphate dehydrogenase/hemoglobin ratio and in the heat stability of the enzyme in hemolysates of ‘old’ and ‘young’ cells. These differences were believed to be due to the aging of the enzyme. As the erythrocyte membrane acts as a molecular sieve under hypotonic conditions [cf. Cseke, E., Varadi, A., Szabolcsi, G., and Biszku. E. (1978) FEBS Lett. 96, 15–18], the hemolysate obtained when a fraction is lysed does not properly represent the content of the lysed cells. As hemoglobin is lost from cells which are not yet lysed, the enzyme/hemoglobin ratio is underestimated in ‘old’ cells and overestimated in ‘young’ cells. It is further shown that the observed differences in the heat stability of glucose‐6‐phosphate dehydrogenase in the fractions obtained by graded hemolysis are due to the presence of different concentrations of endogeneous NADP. Therefore the published data obtained by graded osmotic hemolysis do not prove the assumption that the enzyme is aging during the lifetime of the ery‐ throcyte.
Anticooperative Binding of l‐Tryptophan to Tryptophanyl‐tRNA Synthetase from Beef Pancreas
Authors: GRAVES Pierre‐Vincent , MAZAT Jean‐Pierre , JUGUELIN Héléne , LABOUESSE Julie , LABOUESSE Bernard
Published: March 2005

Equilibrium dialysis and gel filtration studies show that tryptophanyl‐tRNA synthetase from beef pancreas binds two molecules of l‐tryptophan per dimer in an anticooperative way.

The binding of tryptophan ellicits a series of spectroscopic changes in the protein as seen by absorbance, fluorescence and circular dichroism. The molar absorption change of the protein‐tryp‐ tophan system upon formation of the complex is Δɛ292= 10400 ± 1000 M−1 cm−1 per dimer. Taking an initial symmetrical dimeric protein the two dissociation constants for tryptophan at pH 8, 25 C are respectively K1= 2.0 ± 0.5 μM and K2= 10 ± 4 μM. They are respectively K1= 1 ± 0.25 μM and K2= 20 ± 8 μM if one considers a sequenced binding of the two tryptophan molecules.

The dichroic band at 290 nm of the free protein disappears when tryptophan is bound. All observed changes are characteristic of tryptophan perturbation and none of tyrosine perturbation. They all exceed the effect that can be expected from the change in environment of the bound tryptophan molecules and modifications of the tertiary structure of the protein have to be taken into account to explain the observed spectroscopic data.
Purification and Molecular Properties of a New Colicin
Authors: CAVARD Daniele , LAZDUNSKI Claude J.
Published: March 2005

The process of isolation and purification of a new colicin isolated from a Citrohacter strain is described. Escherichia coli sensitive cells are protected by vitamin B12 from the action of this bacteriocin; this suggests that it belongs to the E group of colicins. Therefore, we have called it colicin E4. It has a molecular weight of 56000 and two molecular forms of isoelectric points 9.4 and 8.2 are separated in electrofocusing on polyacrylamide gels. It has a sedimentation coefficient of 3.4 S and the absorption coefficient A1280% is 6.23 cm−1. Using an antibody raised against pure colicin E4, no cross‐reaction was detected against colicins A, E1 or K. The physiological effect of colicin E4 on sensitive cells is very similar to that of colicins El, K or I which disrupt the energized membrane state.
Interaction of Colicin E4 with Specific Receptor Sites Mediates Its Cleavage into Two Fragments Inactive Towards Whole Cells
Authors: CAVARD Daniéle , LAZDUNSKI Claude
Published: March 2005

Colicin E4 has been iodinated with [125l]iodine. The relationship between the degree of iodination and the biological activity has been determined. In contrast to colicins E2, E3, Ia, Ib or cloacin, conventional saturation curves with sensitive cells are not obtained with colicin E4. A turnover ofcolicin on the receptors is evidenced. Analysis of the colicin molecules (Mr 56000 ± 2000) incubated in buffer with sensitive cells, on sodium dodecylsulfate/polyacrylamide gels shows that cleavage of the colicin molecules occurs to yield two fragments of 27000 Mr and 26000 Mr respectively. Strains selected for resistance to colicin E4 are defective in their ability to cleave this bacteriocin, whereas tolerant strains, which have an intact receptor but are not killed by colicin E4. cleave like sensitive cells. Colicin ‐ receptors interaction, which results in cleavage, is much less sensitive to the degree of iodination than the biological activity. Kinetics of cleavage as well as the effect of various treatments of sensitive cells are described. Among the various parameters which alter the cleavage, the most interesting is the ionic strength effect. In the presence of 0.5% or 1% NaCl, colicin E4 cleavage practically is not evidenced. We suggest that the equilibrium constant of the colicin‐receptor complex is very sensitive to ionic strength. The mechanism of colicin E4 cleavage with regard to the killing action is discussed.
Deazaflavins as Cofactors for Flavocytochrome b2 from Baker's Yeast
Authors: POMPON Denis , LEDERER Florence
Published: March 2005

Flavin‐free cytochrome b2 can be reconstituted with 5‐deaza‐5‐carba‐flavin mononucleotide (5‐deaza‐FMN). The resultant enzyme is reduced by lactate and reoxidized by pyruvate, but electrons are not transferred to the heme and no ferricyanide reductase activity can be measured. Tritium transfer experiments from labeled substrate to analog have yielded an isotope discrimination factor of 12. Back transfer (from enzymatically labeled analog to pyruvate) is essentially complete. The affinity of 5‐deaza‐FMN for flavin‐free cytochrome b2 has been found to be similar to that of FMN. Its redox potential is −230 mV. Sulfite forms a reversible covalent adduct with the analog (Kd= 2.8 μM) as it does with the normal cofactor. The reactivity of 2‐hydroxy‐3‐butynoic and 2‐oxo‐3‐butynoic acids toward bound 5‐deaza‐FMN has also been examined; no evidence could be obtained for covalent adduct formation. Difference spectra showed the appearance of a band centered at 423 nm upon binding of oxidized 5‐deaza‐FMN; this band was attributed to an interaction between heme and the analog; it is not observed with the natural cofactor.

In contrast, no evidence was obtained for binding of 1‐deaza‐FMN to flavin‐free cytochrome b2.

A comparison is presented between known data concerning flavocytochrome b2 and lactate oxidase from Mycobactevium smegmatis; they point to a highly similar environment around N‐5, C‐4a and O‐4 of the isoalloxazine ring in the two proteins. It is suggested that the difference between flavin‐dependent dehydrogenases/electron transferases and dehydrogenases/oxidases might be the interaction between flavin and protein in the N‐1‐O‐2 area.
Low‐Molecular‐Weight Substrate for the Lysozyme of T4 Bacteriophage
Authors: BIEŃKOWSKA Krystyna , TAYLOR Alina
Published: March 2005

It has been shown that muropeptide CB, the chemically defined product of Escherichia coli B murein digestion by phage λ endolysin, is the substrate for T4 lysozyme. This is the tetrasaccharide GlcNAc‐MurNAc‐GlcNAc‐anMurNAc in which the carboxyl groups of MurNAc and anMurNAc residues are substituted by tetrapeptide lAla‐dGlu‐msA2pm‐dAla (MurNAc =N‐acetylmuramic acid, GlcNAc =N‐acetyl‐d‐glucosamine, anMurNAc = 1,6‐anhydro‐N‐acetylmuramic acid, lAla =l‐alanine, dClu =d‐glutamic acid, msA2pm =meso‐diaminopimelic acid). The substrate contains one bond hydrolysable by T4 lysozyme. The products of hydrolysis are the easily identifiable disaccharide muropeptides C6 (GlcNAc‐MurNAc‐lAla‐dGlu‐msA2pm‐dAla) and CA (GlcNAc‐anMurNac‐lAla‐dGlu‐msA2pm‐dAla). Thus the substrate may be used for the specific identification of murein N‐acetylmuramoylhydrolases of the T4 lysozyme type, as well as for any quantitative measurement of the enzymatic reaction.
Coupling between Steroid Oxidoreductions in vivo. Deuterium Transfer from [17α2‐2H]Estradiol to C19 Steroids
Authors: CRONHOLM Tomas , RUDQVIST Ulf
Published: March 2005

Female rats were given [17α‐2H]estradiol, either free intraperitoneally or as the 3‐sulphate in intravenous infusion. Metabolites of reducible steroids were isolated from the steroid disulphate fraction obtained from bile, and the deuterium content was analyzed by gas chromatography – mass spectrometry. Deuterium was transferred to the D‐ring of the 17β‐hydroxysteroid formed from intraperitoneally administered 3α‐hydroxy‐5α‐androstan‐17‐one or 3β‐hydroxy‐5α‐androstan‐l7‐one or 5α‐ androstane‐3,17‐dione. The 5α‐androstane‐3β,17β‐diol contained more deuterium (37 – 52% of the estradiol 2H excess) when formed from 3α‐hydroxy‐5α‐androstan‐17‐one than when it was formed from 3β‐hydroxy‐5α‐androstan‐17‐one or 5α‐androstane‐3,17‐dione (7–11% and 12–19%, respectively, of the estradiol 2H excess). Deuterium was also incorporated in the D‐ring of 5α‐androstane‐3β, 17β‐diol formed from 17β‐hydroxy‐4‐androsten‐3‐one, indicating oxidoreduction of the oxygen function at C‐17. No deuterium was found in this metabolite when the 17‐sulphate was administered. Deuterium from [17α‐2H]estradiol 3‐sulphate was incorporated in the steroid diols formed from the intravenously administered 3‐sulphates of 3α‐hydroxy‐ and 3β‐hydroxy‐5α‐androstan‐17‐one. The deuterium content in these steroids diols was 11–45% of the estradiol 2H excess. The results are consistent with an interaction between estradiol metabolism and oxidoreduction of C19 steroids mediated by a common 17β‐hydroxysteroid: NAD(P) oxidoreductase. The transfer of deuterium might thus be due to a slow dissociation of the enzyme‐reduced coenzyme complex. compared to the dissociation and association of the substrates. The difference in degree of transfer to the 17α‐position of 3α‐hydroxy and 3β‐hydroxy steroids is probably explained by the presence of at least two 17β‐hydroxysteroid: NAD(P) oxidoreductases, differing in specificity and kinetic parameters.
Intracellular Localization of Exogenous β‐Glucuronidase in Cultured Skin Fibroblasts
Authors: BACH Gideon , LIEBMANN‐EISENBERG Anette
Published: March 2005

The intracellular localization of exogenously supplied human platelet β‐glucuronidase in cultured skin fibroblasts derived from a β‐glucuronidase‐deficient patient was studied. Four cellular fractions were obtained by differential speed centrifugation. Following two days of incubation, the exogenously supplied enzyme exhibited a distribution pattern identical to that of endogenous β‐hexosaminidase. Disruption of membranes by freezing and thawing caused a 35% increase of the enzyme activity, thus indicating a latent activity following the internalization. This indicated localization in the lysosomal fractions. Longer incubation periods led to an intracellular shift of the engulfed enzyme from the lighter lysosomal fraction to heavier particles. Once located in the heavier fraction, the enzyme was relatively stable, and participated in the catabolism of 35S‐labeled mucopolysaccharides which had accumulated in the lysosomes of these fibroblasts. A marked reduction in the accumulated mucopolysaccharides of the lysosomal fraction was observed following addition of the enzyme. This was accompanied by the formation of smaller sized molecules.
Purification and Characterization of Proinsulin mRNA from Rat B‐Cell Tumor
Authors: ITOH Nobuyuki , NOSE Kiyoshi , OKAMOTO Hiroshi
Published: March 2005

Proinsulin mRNA was purified from rat B‐cell tumor induced by streptozotocin and nicotinamide. The purification was achieved through the use of oligo(dT)‐cellulose chromatography and sucrose gradient centrifugation. As judged by translational activity in a cell‐free protein‐synthesizing system of wheat germ, proinsulin mRNA was purified 362‐fold from total nucleic acid of the B‐cell tumor. Purified proinsulin mRNA migrated as a single symmetrical peak on sucrose gradient centrifugation and as a single band on polyacrylamide gel electrophoresis in the presence of formamide. The translation product of purified proinsulin mRNA in the cell‐free protein‐synthesizing system was found to be only preproinsulin, as analyzed by dodecylsulfate/polyacrylamide gel electrophoresis and immunoprecipitation.

The molecular weight of proinsulin mRNA, determined by polyacrylamide gel electrophoresis in the presence of formamide, was approximately 200000 (equivalent to approximately 570 nucleotides). Almost all of the proinsulin mRNA was bound to oligo(dT)‐cellulose, and the translation of proinsulin mRNA in a cell‐free protein‐synthesizing system of wheat germ was completely inhibited by 7‐methylguanosine 5′‐phosphate. These results indicated that the nucleotide sequences in proinsulin mRNA consisted of 58% translatable sequences and 42 % untranslatable sequences, presumably containing a capped structure, a 7‐methylguanosine residue, at the 5′ terminus and a poly(A) sequence at the 3′ terminus.

The translation product of purified proinsulin mRNA, preproinsulin, was found to consist of two types of preproinsulin I (57%) and preproinsulin II (43%). This result indicated that the isolated proinsulin mRNA consisted of two types of proinsulin I mRNA and proinsulin II mRNA in approximately equal amounts.

Complementary DNA was synthesized with proinsulin mRNA as a template. From the experiment of hybridization kinetics under conditions of excess RNA, the r0t1/2, of proinsulin mRNA was found to be 2.1 × 10−4 mol 1−1 s. This result also confirmed that proinsulin mRNA had been isolated in a high degree of purity.
Ribosome Biosynthesis Is not Necessary for Initiation of DNA Replication
Authors: GRUMMT Friedrich , GRUMMT Ingrid , MAYER Evelyne
Published: March 2005

Synthesis of mature 28‐S ribosomal RNA and 60‐S ribosomal subunits is inhibited in baby hamster kidney (BHK) cell line ts 422E at non‐permissive temperature (39°C). This leads to a 66% decrease of total ribosomes per cell, a marked imbalance between the large and small ribosomal subunits in the cytoplasm and a decrease of cells per dish after prolonged culture at 39°C. However, inhibition of ribosome synthesis does not affect progression of cells through the G1 period of the cell division cycle, the length of the pre‐replicative period, and the rate of entry of cells into S phase. In contrast to culture at non‐permissive temperature, culture of BHK ts 422E cells in the presence of 0.04 μg/ml actinomycin D at 33°C inhibits markedly the entry into S period. It is concluded that low doses of actinomycin D exert their inhibitory effect on cell growth by preventing maturation and transport of mRNA rather than by interfering with ribosome synthesis. Microfluorometric analysis revealed only slight differences in the distribution of BHK ts 422E cells in G1, S and G2 phases of the cycle either when cultured at 33°C or at 39°C. When too few ribosomes per cell are produced in BHK ts 422E cells at 39°C, cells do not seem to be arrested reversibly at a specific point of the cell cycle but rather to die at random.
1H‐Nuclear‐Magnetic‐Resonance Studies on Glycophorin and Its Carbohydrate‐Containing Tryptic Peptides
Authors: EGMOND Maarten R. , WILLIAMS Robert J. P. , WELSH E. Jane , REES David A.
Published: March 2005

The proton nuclear magnetic resonance (1H‐NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of α‐helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met‐81 residue in glycophorin.

Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C‐3 to C‐11 methylene resonances. The bound dodecyl‐sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate‐protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.
Purification and Some Properties of a Hitherto‐Unknown Enzyme Reducing the Carbon‐Carbon Double Bond of α,β‐Unsaturated Carboxylate Anions
Authors: TISCHER Wilhelm , BADER Johann , SIMON Helmut
Published: March 2005

2‐Enoate‐reductase, a previously unknown soluble enzyme is present in Clostridium kluyveri and another Clostridium species growing on (E)‐2‐butenoate. From the latter the reductase was purified 88‐fold with an overall yield up to 74%. The enzyme was pure as judged by polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate as well as by isoelectric focusing.

The purification of the enzyme was performed in the presence of (E)‐2‐methyl‐2‐butenoate as substrate to keep the enzyme in the oxidized state and under anaerobic conditions. The purification procedure included an ammonium sulphate precipitation, chromatography on DEAE‐Sepharose CL‐6B, hydroxylapatite and Sepharose CL‐6B.

The enzyme reduces different α,β‐unsaturated carboxylate anions such as (E)‐2‐butenoate, (E)‐2‐methyl‐2‐butenoate, (E)‐cinnamate and probably many others in a NADH‐dependent reaction to the saturated carboxylate anions. Fumarate, 3‐phenyl‐2‐propinate, 2‐enoyl‐methyl and CoA esters proved not to be substrates for the purified reductase. NADPH does not act as an electron donor.

The enzyme was shown to have a molecular weight of about 450000 by gel chromatography. It consists of subunits with a molecular weight of 78000. Per subunit about 1 FAD, 3.5–3.8 atoms of iron and 4.0 labile sulphur atoms have been found indicating a conjugated iron‐sulphur flavoprotein. Copper could not be detected. The isoelectric point was 8.4.

As shown by absorption spectroscopy the enzyme can be reduced by NADH and reoxidized with dichloroindophenol, hexacyanoferrate III, oxygen and substrates.

Addition of 8 mol p‐hydroxymercuribenzoate to 1 mol subunit completely destroyed the activity of the reductase.

So far no physiological role of the enzyme is known.
Outer Membrane Proteins of Smooth and Rough Strains ofProteus mirabilis
Authors: ROTTEM Shlomo , MARKOWITZ Ora , HASIN Miriam , RAZIN Shmuel
Published: March 2005

The outer membranes of the smoothProteus mirabilisS1959 strain and its rough R13, R110, R51 and R45 mutants were isolated by sonication of the cells and sucrose density gradient centrifugation. The outer membrane of the rough strains had a lower density than that of their parent smooth strain, but the protein‐to‐phospholipid ratios were the same. The electrophoretic patterns of outer membrane polypeptides of the S and R strains in sodium dodecylsulfate/polyacrylamide gels were identical, with two major polypeptide bands, C1and C2(Mr39000 and 38000) predominating. The C1polypeptide band was a heat‐modifiable polypeptide, which migrated as a band atMr, 33000 when membranes were solubilized at 37°C or 50°C, and atMr39000 when solubilization was at 100°C. Susceptibility of outer membrane polypeptides to proteolytic digestion was found to be higher in isolated outer membrane preparations of the rough strains than in the smooth strain, suggesting that the availability of the polypeptide chains to proteolytic activity depends on the length of the polysaccharide chains of the outer membrane lipopolysaccharide.
Regulation of Glycogen Synthesis in Rat‐Hepatocyte Cultures by Glucose, Insulin and Glucocorticoids
Authors: SCHUDT Christian , Tilch Karin , Bley Peter , Toledo Avi
Published: March 2005

The changes in glycogen content and in its rate of synthesis in two‐day‐old primary cultures of rat hepatocytes were assessed under various conditions. Hepatocytes cultivated in serum‐free and hormone‐free medium switch from glycogen degradation to glycogen deposition at 10.3 mM glucose. After pretreatment of the cells with glucocorticoids this threshold was reduced, in the absence or presence of insulin, to 5.4 or 2.1 mM glucose, respectively. The rate of glycogen synthesis in the presence of 10 mM glucose was amplified from 5nmol × h−1× mg protein−1 to 20 nmol glucose × h−1× mg protein−1 after pretreatment with triamcinolone. Glucagon pretreatment also significantly increased the subsequent glycogen synthesis rate. Insulin addition accelerated glycogen synthesis about twofold regardless of the pretreatment. The dose‐response relationship between insulin concentration and glycogen synthesis rate showed half‐maximal effect at 0.62 ± 0.22 nM (mean ± S.D.) insulin. Pretreatment of hepatocytes with glucocorticoids, glucagon, insulin or combinations of these hormones did not significantly change the concentration which gives the half‐maximal effect.
External and Internal Forms of Yeast Aminopeptidase II
Authors: FREY Jürgen , RÖHM Klaus‐Heinrich
Published: March 2005

Intact cells of Saccharomyces cerevisiae catalyze the hydrolysis of various aminopeptidase substrates. This activity is not due to permeation of substrates and products but exerted by an external enzyme.

From its substrate specificity and the effects of pH and inhibitors the enzyme was identified as aminopeptidase II.

About 40% of total aminopeptidase II activity is detectable with untreated exponentially growing cells. Up to two thirds of the external enzyme is released into the medium during enzymic digestion of the cell wall, while little enzyme is liberated by osmotic shock. Membrane preparations contained only small amounts of aminopeptidase II; thus, the localization of the external enzyme appears to be similar to that of the so‐called ‘periplasmic’ yeast hydrolases.

By cytochemical methods the presence of aminopeptidase II in the cell envelope was visualized.

In contrast to aminopeptidase II, yeast dipeptidase is an entirely intracellular enzyme.
Trans‐membrane Translocation of Proteins
Authors: VON HEIJNE Gunnar , BLOMBERG Clas
Published: March 2005

As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physicochemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans‐membrane proteins.
Partial Purification and Characterization of the Proteins from the 40‐S Ribosomes of Artemia salina and Wheat Germ
Authors: TING SHIH Chao‐Yun , TOIVONEN John E. , CRAVEN Gary R.
Published: March 2005

The proteins were extracted from purified 40‐S ribosomes derived from wheat germ and Artemia salina and separated by carboxymethylcellulose ion‐exchange chromatography. Approximately four proteins from Artemia and four proteins from wheat germ were separated in a state of high purity. All proteins were identified by co‐electrophoresis using a two‐dimensional polyacrylamide gel system. A total of 30 unique proteins were found for Artemia and 32 proteins for wheat. The molecular weights of all proteins were estimated by sodium dodecylsulfate gel electrophoresis. Assuming each protein to be present in one copy per 40‐S ribosome, the total protein molecular weight was estimated to be 560000 associated with Artemia 40‐S particles and 550000 associated with wheat germ 40‐S ribosomes.
Effect of Sulphated Glycopeptides on Kinetics of 3‐OMethyl Glucose and 2‐Deoxyglucose Transport by Epithelial Cells Isolated from Rabbit Small Intestine
Authors: MIAN Nasi , ANDERSON Caroline E. , KENT Paul W.
Published: March 2005

Suspensions of cells isolated from rabbit small intestine were prepared which showed a considerable rate of respiration after 4 h of their isolation. Rabbit intestinal cells accumulated 3‐O‐methyl‐glucose by an active transport mechanism which was Na+‐dependent whereas 2‐deoxyglucose was transported by a saturable pathway. Analysis of the initial transport data indicated that the uptake of both sugars by cells followed Michaelis‐Menten‐type kinetics. These cells also showed a considerable degree of 2‐deoxyglucose phosphorylation and the transport of the sugar into the cells appeared to be a rate‐limiting factor for its phosphorylation.

Sulphated glycopeptide had no effect on the respiration rate of these cells but it increased the transport of 3‐O‐methyl‐glucose and 2‐deoxyglucose into cells by many fold compared with untreated cells. Kinetic analysis of the data indicated that the increase in the transport was due to increased V while the Km remained unchanged. Presence of sulphated glycopeptide had no effect on the kinetics of phosphorylation of 2‐deoxyglucose. These effects of sulphated glycopeptide on the transport activity of the cells have been discussed as a result of an increase in the number of sugar sites in the cell membrane due to unmasking or activation of preexisting sites owing to the interaction between the cell membrane components and highly polyanionic sulphated glycopeptides.
N‐Acetylaminoacyl‐p‐Nitranilidase from Human Placenta
Authors: UNGER Thomas , NAGELSCHMIDT Manfred , STRUCK Hansjürgen
Published: March 2005

An enzyme hydrolyzing N‐acetylaminoacyl‐p‐nitroanilides has been isolated from mature human placentae by a six‐step procedure comprising extraction from a placenta homogenate, ammonium sulfate fractionation, treatment with isopentyl alcohol, chromatography on CM‐Sephadex, protamine sulfate precipitation and gel filtration on an Ultrogel AcA 34 column. About 2500‐fold enrichment was achieved from placenta homogenate. The purified enzyme preparation showed a single band on polyacrylamide disc electrophoresis. The molecular weight was estimated to be 380000 by gel filtration. Placental extracts contain two main isoenzymes of pI 3.9 and 4.5 respectively.

Activity was strongly inhibited by chloromercuribenzoate, slightly inhibited by Ca2+ and cysteine; no activation could be detected. The enzyme exhibits an exopeptidase‐like activity towards acetyl‐dipeptides with a certain specifity towards N‐acetylalanyl‐alanine; N‐acetylalanine‐p‐nitroanilide, however, is hydrolyzed four times faster.

With N‐acetylalanine‐p‐nitroanilide as substrate the pH optimum was 8.0–8.2; Km was 2.13 mmol/l. N‐Acetylleucine‐p‐nitroanilide and N‐acetyltyrosine‐p‐nitroanilide were split slowly; N‐acetylalanyl‐alanyl‐alanine‐p‐nitroanilide, N‐butyloxycarbonyl‐alanyl‐alanine‐p‐nitroanilide, un‐substituted analogous aminoacyl‐p‐nitroanilides and several protein substrates were not hydrolyzed.

The functions of the enzyme are still unknown.
Methyl Acceptors for Protein Methylase II from Human‐Erythrocyte Membrane
Authors: GALLETTI Patrizia , PAIK Woon Ki , KIM Sangduk
Published: March 2005

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S‐adenosylmethionine: protein‐carboxyl O‐methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff‐2 and NaIO4/Schiff‐1 positive bands) and ‘band 4.5’ were identified as two major classes of methyl‐acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, ‘band 4.5’ was the only major methyl‐acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 μM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.
Ionized and Bound Calcium Inside Isolated Sarcoplasmic Reticulum of Skeletal Muscle and Its Significance in Phosphorylation of Adenosine Triphosphatase by Orthophosphate
Authors: PRAGER Rudolf , PUNZENGRUBER Christian , KOLASSA Norbert , WINKLER Franz , SUKO Josef
Published: March 2005

Calcium loading of skeletal muscle sarcoplasmic reticulum performed passively by incubation with high calcium concentrations (0.5–15 mM) on ice gives calcium loads of 50–60 nmol/mg sarcoplasmic reticulum protein. This accumulated calcium is not released by EGTA [ethyleneglycol bis‐(2‐aminoethyl)‐N, N, N′, N′‐tetraacetic acid], but almost completely released by ionophore X‐537A plus EGTA or phospholipase A plus EGTA treatment and is therefore assumed to be inside the sarcoplasmic reticulum. This calcium is distributed in one saturable and one non‐saturable calcium compartment, as derived from the dependence of the calcium load on the calcium concentration in the medium. These compartments are assigned to bound and ionized calcium inside the sarcoplasmic reticulum, respectively. Maximum calcium binding under these conditions was 33 nmol/mg protein with an apparent half‐saturation constant of 5.8 nmol/mg free calcium inside, or between 1.2 and 0.6 mM free calcium inside, assuming an average vesicular water space of 5 or 10 μl/mg protein respectively.

Calcium‐dependent phosphorylation of sarcoplasmic reticulum calcium‐transport ATPase from orthophosphate depends on the square of free calcium inside, whilst inhibition of phosphorylation depends on the square of free calcium in the medium. Calcium‐dependent phosphorylation appears to be determined by the free calcium concentrations inside or outside allowing calcium binding to the ATPase according to the two classes of calcium binding constants for low affinity calcium binding or high affinity calcium binding, respectively.

It is further suggested that the saturation of the low‐affinity calcium‐binding sites of the ATPase facing the inside of the sarcoplasmic reticulum membrane is responsible for the greater apparent orthophosphate and magnesium affinity in calcium‐dependent phosphorylation than in calcium‐independent phosphorylation from orthophosphate. Maximum calcium‐dependent phosphoprotein formation at 20°C and pH 7.0 is about 4 nmol/mg sarcoplasmic reticulum protein.
Purification and Properties of RNA‐Dependent DNA Polymerase from Cytoplasmic A‐Type Particles of Murine Mammary Tumor Virus
Authors: KOHNO Michiaki , ISHIHAMA Akira
Published: March 2005

The RNA‐dependent DNA polymerase associated with cytoplasmic A‐type particles of murine mammary tumor virus was isolated to near homogeneity by a procedure which includes dissociation of proteins from RNA by centrifugation in a step gradient of cesium chloride, followed by an affinity chromatography on poly(rC)‐agarose column. Two species of DNA polymerase were separated by the chromatography: enzyme I in 0.55 M NaCl and enzyme II in 0.80 M NaCl eluate, respectively.

The purified DNA polymerases consist of two major polypeptides, with molecular weights of 94000 and 42000, as the intrinsic subunits. Both enzyme protomers with a sedimentation coefficient of 6.3–6.4 S and a molecular weight of 115000–120000 associate to form active oligomers in low‐ionic‐strength buffer.

Both enzymes catalyzed the hydrolysis of RNA in RNA. DNA hybrids as well as the RNA‐dependent synthesis of DNA; these are the intrinsic activities of the reverse transcriptase from B‐type particles of murine mammary tumor virus as well as from avian and mammalian C‐type oncornaviruses. The general catalytic properties are similar to those of the enzyme from B‐type particles. Compared with DNA polymerases I, DPU'A polymerase II exhibited a high affinity for all the template‐primers tested and, in addition, a high preference for (rC)n· (dG)12–18.
Turnover Rates of Hexokinase I, Phosphofructokinase, Pyruvate Kinase and Creatine Kinase in Slow‐Twitch Soleus Muscle and Heart of the Rabbit
Authors: ILLG Dieter , PETTE Dirk
Published: March 2005

Hexokinase I was purified from rabbit heart to a specific activity of 70 U/mg protein. The purified enzyme was electrophoretically homogeneous with an apparent molecular weight of 102000. Purified immunoglobulins from sheep were used to titrate the percentage of hexokinase I in various tissues of the rabbit. Precipitating antibodies from sheep were also prepared against rabbit muscle MM‐creatine kinase, phosphofructokinase and pyruvate kinase. Appararent turnover rates of these phosphotransferases and of hexokinase I were determined in rabbit heart and soleus muscle by means of the immunoprecipitation technique after single pulse labelling with [U‐14Cl]leucine in vivo. Apparent half‐lives of phosphofructokinase, pyruvate kinase and hexokinase I were 0.56 d, 0.73 d and 0.93 d in rabbit heart. In slow‐twitch soleus muscle half‐lives of phosphofructokinase, pyruvate kinase, hexokinase I and creatine kinase were 0.63 d, 0.72 d, 0.85 d and 0.82 d. The similarity of the rate constants of degradation of these enzymes is interpreted as an indication that different tissue concentrations result primarily from different rates of synthesis.
Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria
Authors: SMITH Colleen M. , PLAUT Gerhard W. E.
Published: March 2005

The contributions of NAD‐specific and NADP‐specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using dl‐threo‐α‐methylisocitrate (3‐hydroxy‐1,2,3–butanetricarboxylate) to specifically inhibit flux through NADP‐specific isocitrate dehydrogenase.

Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20–40% by inhibition of NADP‐isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added dl‐isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite α‐methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of α‐methylisocitrate (400 μM) sufficient for 99.99%, inhibition of NADP‐isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 ±6% and 19 ± 11% (mean ± SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3–4‐fold, decreased NADPH levels by 30–40% and 2‐oxoglutarate accumulation by about 40%. The further addition of 600 μM α‐methylisocitrate decreased the NH4Cl‐stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and α‐methylisocitrate as in their absence.

Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD‐specific 3–hydroxybutyrate dehydrogenase, 85–100% of the rate of 3‐hydroxybutyrate formation was retained in the presence of 400–900 μM α‐methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite α‐methylisocitrate concentration was from 20–40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite α‐methylisocitrate concentration was about 60% of the uninhibited rate.
Intracellular Enzymes of Collagen Biosynthesis in 3T6 Fibroblasts and Chick‐Embryo Tendon and Cartilage Cells
Authors: RISTELI Leila , RISTELI Juha , SALO Lüsa , KIVIRIKKO Kari I.
Published: March 2005

An increase of about 4–9‐fold was found in prolyl and lysyl hydroxylase activities per cell when 3T6 fibroblasts were grown from the early‐log to the stationary phase, whereas essentially no changes were found in hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyl‐transferase activities. A control experiment using a different assay procedure indicated that the increase in prolyl hydroxylase activity was not due to any methodological artefact. The results demonstrate for the first time that these four intracellular enzymes of collagen biosynthesis are not regulated in an identical manner within one cell type.

Freshly isolated chick embryo cartilage cells, which synthesize type I1 collagen, had higher lysyl hydroxylase and hydroxylysyl galactosyltransferase activities than freshly isolated chick embryo tendon cells, whereas no difference was found between them in prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activities. The greater extent of the hydroxylation of lysyl residues and galactosylation of hydroxylysyl residues in type I1 than in type I collagen is thus probably due in part to a higher activity of the corresponding enzymes in the cartilage cells, but the greater extent of the glucosylation of galactosylhydroxylysyl residues cannot be explained by any higher activity of the glucosyltransferase.

Prolyl and lysyl hydroxylase activities were markedly lower in the cultured 3T6 cells than in freshly isolated chick embryo tendon cells, particularly when expressed per unit solubilized cell protein. These data agree with the lower rate of collagen production by the 3T6 cells. Unexpectedly, the two hydroxylysyl glycosyltaransferase activities were somewhat higher in the 3T6 cells than in the tendon cells when expressed per cell, a finding which may explain the pronounced glycosylation of the collagen synthesized by the 3T6 cells.

On incubation of the 3T6 cells with 5 μg/ml puromycin for 10 h the intracellular enzyme activities of collagen biosynthesis decreased by about 15–30% in the early‐log phase and by about 10–20% in the late‐log phase. The results suggest that the half‐lives of all four enzyme activities are at least about 24 h in the early‐log phase and possibly even longer in the late‐log phase.
Papulacandin B: an Inhibitor of Glucan Synthesis in Yeast Spheroplasts
Authors: BAGULEY Bruce C. , RÖMMELE Günter , GRUNER Johannes , WEHRLI Walter
Published: March 2005

Papulacandin B, the main component of a series of antibiotics produced by the deuteromycete Papularia sphaerosperma, inhibits the growth of yeasts. It is a highly amphophilic substance containing residues of glucose, galactose and two long‐chain unsaturated fatty acids. It does not cause the release of potassium ions from yeast cells and therefore differs in its mode of action from the polyene antibiotics. Papulacandin B, at concentrations slightly below the minimal inhibitory concentration, partially but selectively inhibited the incorporation of glucose into cells of Saccharomyces cerevisiae and Candida albicans. A much clearer effect was observed on spheroplasts, obtained by digestion of the yeast cell wall with snail digestive enzyme. Incubation of spheroplasts in a minimal medium stabilised by sorbitol led to the incorporation of glucose into alkali‐insoluble glucan, mannan and a fraction containing mainly alkali‐soluble 1,3‐glucan together with some glycogen. Papulacandin B inhibited the synthesis of the alkali‐insoluble fraction, while causing a slight stimulation of glucose incorporation into the other two polysaccharide fractions. The 50% inhibitory concentrations of papulacandin B for glucan synthesis in S. cerevisiae spheroplasts and C. albicans spheroplasts were respectively 0.16 μg/ml and 0.03 μg/ml. C. albicans cells were irradiated with ultraviolet light and selected for maximum resistance to papulacandin B. The 50% inhibitory concentration for glucan synthesis in spheroplasts prepared from this mutant was 2.5 μg/ml. Echinocandin B, a polypeptide antibiotic containing a long‐chain fatty acid, also inhibited the synthesis of glucan in spheroplasts. It is concluded that papulacandin B and probably also echinocandin B inhibit glucan synthesis during cell‐wall synthesis, and thus cause lysis of cells by osmotic rupture.
Binding of Antibodies against Histone H1 to Unfolded and Folded Nucleofilaments
Authors: TAKAHASHI Kei , TASHIRO Yutaka
Published: March 2005

Rabbit antibodies to calf thymus histone H1 were purified by affinity chromatography on histone‐H1–Sepharose and used as a probe for detecting histone H1 in the nucleofilaments prepared from rat liver nuclei. Binding of the antibodies to the unfolded form of nucleofilaments in 5 mM NaCl and to the folded form in 80 mM NaCl was compared. Sucrose density gradient analyses clearly show that the antibodies can preferentially bind to nucleofilaments in 5 mM NaCl but not in 80 mM NaCl. The antibodies, however, can bind to mononucleosomes in 80 mM NaCl. These results suggest that antigenic determinants of histone H1 in unfolded nucleofilaments and in mononucleosomes are accessible to the antibodies, while those in folded nucleofilaments are not. This is consistent with the view that histone H1 is used for folding and packing of a nucleosomal chain.
On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids
Authors: LAANE Colja , HAAKER Huub , VEEGER Cees
Published: March 2005

The effect of O2 on the rate of ATP synthesis and the P/O ratio has been studied in membrane vesicles of Azotobacter vinelandii and Rhizobium leguminosarum bacteroids (strain PRE). It was observed that the P/O ratio rises to a maximum value when the O2 input is increased, but declines when O2 becomes detectable in the medium.

Evidence is presented, that in A. vinelandii H2 donates its electrons at a fourth proton‐translocating site. The presence and the efficiency of this site is independent of the growth conditions and it appeared that no flavoproteins are directly involved in the oxidation of H2. The unidirectional hydrogenase in membrane vesicles of A. vinelandii is not inhibited by acetylene. The function of hydrogenase in the process of nitrogen fixation by A. vinelandii is discussed.

Two types of membrane vesicles of R. leguminosarum bacteroids were prepared. Membranes isolated in a low salt medium lose the ability to couple succinate oxidation to oxidative phosphorylation; upon isolation in a high salt medium this ability is preserved. In contrast the efficiency of oxidative phosphorylation with NADH is not affected by the isolation procedure. The efficiency of energy coupling in membranes vesicles of R. leguminosarum bacteroids can be enhanced by the addition of fatty‐acid‐free bovine serum albumin. No active membrane‐bound hydrogenase could be detected in membrane vesicles of R. leguminosarum bacteroids.
Crosslinking Studies with the Pyruvate Dehydrogenase Complexes from Azotobacter vinelandii and Escherichia coli
Authors: DE ABREU Ronney A. , DE VRIES Joop , DE KOK Arie , VEEGER Cees
Published: March 2005

The spatial arrangements of the individual components of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli have been studied by crosslinking the complex with bis(imido esters) of different chain lengths and with dimethyl‐3,3′‐dithiobis(propionimidate), a bis(imido ester) containing a cleavable disulfide linkage between the two reactive groups. The three‐component complexes of A. vinelandii and E. coli show almost identical crosslink patterns. These indicate that each component exists in at least a dimeric structure and that all three components contact each other. In contrast, in the four‐component complex from A. vinelandii heterologous dimer formation was only found between lipoamide dehydrogenase and high‐molecular‐weight trans‐acetylase and between pyruvate dehydrogenase and low‐molecular‐weight transacetylase. A model is presented to account for these observations.

In the presence of pyruvate, Mg2+ and thiamin diphosphate extensive crosslinking with bis‐(imido esters) takes place between the transacetylase chains of the E. coli complex and also between the high‐molecular‐weight transacetylase chains of the A. vinelandii complex. This indicates a conformational change of the transacetylase core induced by acetylation of this component, exposing lysyl residues previously hidden.

Crosslinking with N,N′‐o‐phenylene‐bismaleimide of the four‐component A. vinelandii complex, pretreated with N‐ethylmaleimide, in the presence of pyruvate, Mg2+ and thiamin diphosphate shows a close positioning of the −SH groups generated by reductive acetylation of the transacetylase isoenzymes (less than 0.6 nm). With the E. coli complex most of the material thus cross‐linked consists of higher polymers with a concomittant decrease of transacetylase monomers. No homologous dimer formation was observed.