bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Cyclic AMP‐Binding Proteins: Inverse Relationship with Estrogen‐Receptors in Hormone‐Dependent Mammary Tumor Regression
Authors: CHO‐CHUNG Yoon Sang , BODWIN Jeffrey S. , CLAIR Timothy
Published: March 2005

Dimethylbenzanthracene‐induced rat mammary carcinomas possess activities binding cyclic adenosine 3′:5′‐monophosphate (cAMP) and estrogen. When dimethylbenzanthracene‐induced tumors regress after ovariectomy of the host, a change in the specific binding of cAMP and estrogen occurs in the tumors. Six days after ovariectomy, cAMP binding increases 5‐fold in the nuclei and 2‐fold in the cytosol of tumors, while nuclear and cytoplasmic estrogen binding decreases by 80% and 50%, respectively. These changes in activities binding cAMP and estrogen are detectable within 1 day after ovariectomy and the changes are reversed when resumption of tumor growth is induced by the injection of 17β‐estradiol. When dimethylbenzanthracene‐induced tumors fail to regress after ovariectomy, the change in activities binding cAMP and estrogen does not occur. Significant increases in the cAMP level as well as in adenylate cyclase and cAMP‐phosphodiesterase activities are also found in the regressing tumors. Concomitant with the increase of cAMP‐binding activity is an increase in histone kinase activity in the regressing tumor. These data suggest the involvement of cAMP in the growth control of a hormone‐dependent mammary tumor.
A 1H Nuclear‐Magnetic‐Resonance Study of the Conformation and the Molecular Dynamics of the Glycoprotein Cow‐Colostrum Trypsin Inhibitor
Authors: WAGNER Gerhard , WÜTHRICH Kurt , TSCHESCHE Harald
Published: March 2005

The glycoprotein cow colostrum trypsin inhibitor was investigated by high resolution 1H nuclear magnetic resonance (NMR) at 360 MHz and, on the basis of the NMR data, compared with the basic pancreatic trypsin inhibitor (Kunitz) from bovine tissue. Detailed studies of the chemical shifts and the exchange kinetics of the labile protons indicated extensive homologies between the spatial structures of the polypeptide chains in the two compounds. This was further corroborated by comparison of the NMR spectral features and the dynamic properties of the aromatic amino acid residues in the two inhibitors. It thus appears that the covalently attached carbohydrate moiety in the colostrum inhibitor has only very limited effects on the spatial structure of the protein part of the molecule. On the other hand, as evidenced by the NMR line widths, the carbohydrate attachment causes a pronounced restriction of the overall mobility of the molecule, indicating a sizeable increase of the average radius of gyration as compared to the basic pancreatic trypsin inhibitor. Possible spatial arrangements of the globular polypeptide and the carbohydrate moieties in the colostrum inhibitor, which would be compatible with the experimental observations, are discussed.
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.
Calcium as Modulator of the Hormonal‐Receptors– Biological‐Response Coupling System
Authors: BONNE Dominique , BELHADJ Omrane , COHEN Paul
Published: March 2005

Activation of 2‐deoxyglucose transport in isolated rat fat cells by insulin is dependent upon the presence of Ca2+ in the external medium. When calcium concentration is kept below 100 μM, insulin acts like a partial agonist, giving only half of the maximal activation obtained normally with a millimolar concentration of this ion. Ocytocin, whose insulin‐like action on adipocytes activates glucose oxidation by these cells, was found to be unable to affect the rate of 2‐deoxyglucose transport. This, together with previous observations, suggests that calcium ions play a role in the mechanism of insulin action possibly by binding selectively to membrane sites involved in the transmission of the hormonal message to the glucose carrier. Ocytocin seems to trigger only intracellular glucose metabolism and it appears that there is an absolute requirement for calcium ions in the activation of a still unknown membrane signal.
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.
Nucleotide Sequence of the Restriction Fragment Hind‐F‐EcoRI1 of Simian‐Virus‐40 DNA (Part of the VP1 Gene)
Authors: CONTRERAS Roland , VAN DE VOORDE André , FIERS Walter
Published: March 2005

The nucleotide sequence of the simian virus 40 (SV 40) genome region between the cleavage sites for restriction endonucleases EcoRI (map position 0) and HindII (map position 0.05) has been determined mainly by the partial chemical DNA degradation procedure of Maxam and Gilbert. This fragment represents 5.3% of the genome of SV 40 and is located in the late region, internally in the VP1 gene. The message strand shows only one open reading frame for translation into protein, which connects to the one for the preceding fragment. On this basis part of the amino acid sequence of the VP1 protein is presented.
Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split
Authors: QUAST Ulrich , ENGEL Jürgen , STEFFEN Erna , TSCHESCHE Harald , KUPFER Sigrid
Published: March 2005

Modified trypsin kallikrein inhibitor (I*), with the reactive‐site peptide bond Lys‐15–Ala‐16 split, reacts with α‐chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C: E + I*⇌ X → C. Formation of X constitutes a fast pre‐equilibrium (equilibrium constant Kx= 7 × 10−5 M. association rate constant kx= 4 × 103 M−1 s−1) to the slow reaction X → C (rate constant kc= 2 × 10−3 s−1), all values at pH 7.5. No intermediate X is observed when α‐chymotrypsin reacts with I*‐OMe in which the carboxyl group of Lys‐15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*‐OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with β‐trypsin and I* or I*‐OMe.
Multi‐Enzyme Complex of Glutamine‐Dependent Carbamoyl‐Phosphate Synthetase with Aspartate Carbamoyltransferase and Dihydroorotase from Rat Ascites‐Hepatoma Cells
Authors: MORI Masataka , TATIBANA Masamiti
Published: March 2005

Glutamine‐dependent carbamoyl‐phosphate synthetase, the first enzyme of de novo pyrimidine nucleotide biosynthesis, was purified 1300‐fold from Yoshida ascites hepatoma cells (AH 13) as a multi‐enzyme complex with aspartate carbamoyltransferase and dihydroorotase, using dimethyl sulfoxide, glycerol and dithiothreitol as enzyme stabilizers. The ratios of the three enzyme activities were initially 1:65:9.2 and were essentially constant throughout the purification, and the final fraction had ratios of 1:37:11 (dihydroorotase assayed in the forward direction). The purified complex had a specific activity of the synthetase of 0.51 μmol × min−1× (mg protein)−1. It was essentially homogeneous on agarose/acrylamide composite gel electrophoresis, analytical ultra‐centrifugation, and electrofocusing; it had an apparent pI of 5.5. The complex had a sedimentation coefficient (s20, w) of 24.6 S in the presence of 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol, and its molecular weight was estimated to be 870000 by sedimentation equilibrium studies.

On polyacrylamide gel electrophoresis in sodium dodecyl sulfate the complex migrated as a single protein band with a molecular weight of 210000, indicating that the complex is composed of four to five subunits of similar size. Sucrose gradient centrifugation in the presence of 1.5 M sodium chloride partially dissociated the three enzyme activities. Limited proteolysis of the complex with pancreatic elastase caused its dissociation and made separable the three activities. The synthetase was recovered as a 197000‐molecular‐weight fragment and the carbamoyltransferase as a 145000‐molecular‐weight fragment. Dihydroorotase was initially recovered as a 197000‐molecular‐weight fragment and was further converted to a 43000‐molecular‐weight fragment. It appears difficult to reconcile these results with the view that the three enzyme activities are located on the same polypeptide chain.
Cytochrome c from Schizosaccharomyces pombe
Authors: SIMON‐BECAM Anne‐Marie , CLAISSE Maurice , LEDERER Florence
Published: March 2005

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four‐residue amino‐terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one‐residue deletion at the C‐terminal end.

After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S‐carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.
Biosynthesis of Thromboxane B2 and 12‐l‐Hydroxy‐5,8,10‐heptadecatrienoic Acid in Human Platelets
Authors: RAZ Amiram , AHARONY David , KENIG‐WAKSHAL Rina
Published: March 2005

Human platelet microsomes convert prostaglandin H2 to thromboxane B2 and 12‐l‐hydroxy‐5,8,10‐heptadecatrienoic acid (12OH‐17:3) in approximately equimolar amounts. The synthesizing activities of both products appear to go in parallel, both activities gradually decline upon storage and are equally destroyed by heat inactivation. Furthermore imidazole, a potent inhibitor of thromboxane synthetase activity, is an equally effective inhibitor of 12OH‐17:3 formation in platelets. These results suggest that both thromboxane B2 and 12OH‐17:3 are derived from a common intermediate.

We propose two alternative pathways for the conversion of prostaglandin H2 to thromboxane A2 and 12OH‐17:3 in human platelets. The first pathway depicts thromboxane A2 as the common intermediate for the formation of both thromboxane B2 and 12OH‐17:3. In the second pathway, prostaglanding H2 is converted to an activated intermediate which is converted to either thromboxane A2 or 12OH‐17:3.
Binding of Microtubule Proteins to DNA: Specificity of the Interaction
Authors: CORCES Victor G. , SALAS José , SALAS María L. , AVILA Jesús
Published: March 2005

Tubulin is detected among the DNA‐binding proteins when an extract from fibroblasts is chromatographed on DNA‐cellulose. Further purification of the colchicine‐binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule‐associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin‐free microtubule‐associated proteins bind to DNA, while tubulin free of microtubule‐associated proteins does not. Microtubule‐associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction.
Localization of the Oxytocin Receptor in the Plasma Membrane of Rat Myometrium
Authors: CRANKSHAW Denis J. , BRANDA Luis A. , MATLIB Mohammed A. , DANIEL Edwin E.
Published: March 2005

The distribution of [3H]oxytocin binding sites among various subcellular fractions of rat myometrium paralleled the distribution of 5′‐nucleotidase, a plasma membrane marker enzyme, but not of NADPH‐cytochrome c reductase or succinate‐cytochrome c reductase, which are endoplasmic reticulum and mitochondrial marker enzymes respectively. [3H]Oxytocin binding to the most enriched plasma membrane fraction showed the degree of selectivity with respect to hormone analogues that is expected for the oxytocin receptor. The binding of oxytocin to this fraction showed an apparent Kd of 1.98 × 10−9 M and a capacity of 1.28 pmol mg−1. It is concluded that the oxytocin receptor is located on the plasma membrane of the smooth muscle cells of the rat uterus.
The Acceptor for Polar Head Groups of the Lipid A Component of Salmonella Lipopolysaccharides
Authors: LEHMANN Volker , REDMOND John , EGAN Aubrey , MINNER Inge
Published: March 2005

We describe here experiments which determine at which stage in the lipid A biosynthesis the polar head groups 4‐aminoarabinose, phosphorylethanolamine and 3‐deoxy‐d‐manno‐octulosonic acid are transferred to the diphosphorylated glucosamine backbone of the lipid A structure. Use was made of a conditional lethal mutant of Salmonella typhimurium (Ts1) which is defective in the synthesis of 3‐deoxy‐d‐manno‐octulosonic acid 8‐phosphate and accumulates under nonpermissive conditions an underacylated lipid A intermediate [Lehmann, Rupprecht and Osborn (1977) Eur. J. Biochem. 76, 41–49]. Pulse‐chase experiments, including a detailed analysis of radioactive pulse and chase products, demonstrated that this underacylated compound is a key intermediate in the lipid A synthesis. It can serve as direct acceptor for the incorporation of the polar head groups 4‐aminoarabinose, phosphorylethanolamine and 3‐deoxy‐d‐manno‐octulosonic acid.

On the basis of these findings some steps in the sequence of reactions involved in the lipid A biosynthesis are proposed.
A Barley Endonuclease Specific for Apurinic DNA
Authors: ŠVACHULOVÁ Jirna , ŠATAVA Jan , VELEMINSKÝ Jiri
Published: March 2005

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation. ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G‐150 chromatography. Purityo of the resulting enzyme was determined by gel electrophoresis and gel chromatogrphy and specificity be testing the activity on intact and depurinated bacterial DNAs. At lower concentrations. the enxyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non‐specific manner. the nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.
Hormonal Regulation of Phosphoenolpyruvate Carboxykinase in Primary Cultures of Adult‐Rat Liver Parenchymal Cells
Authors: OLIVER Ivan T. , EDWARDS Anthony M. , PITOT Henry C.
Published: March 2005

Adult rat liver parenchymal cells, maintained in primary monolayer culture on floating collagen gels, were used for studies on the hormonal regulation of phosphoenolyruvate carboxykinse. In cells maintained in a defined, serum‐free medium containing insulin but no other hormones, phosphoenolpyruvate carboxykinse activity expressed on the basis of cell DNA content decreased over 3 days in culture with a half‐life of 9–10 h. Treatment of cells with glucagon or N6, O2′‐dibutyryladenosine 3′:5′ monophosphate (Bt2‐cAMP) caused an increase in enxyme activity up to 4‐fold higher than in untreated cells. There was little or no response during the first 24 h of culture but addition of either glucagon or Bt2‐cAMP after this period resulted in significant enzyme induction within 3 h. When cells were maintained for 2–3 days after attachment directly to palstic culture dishes rather than to collagen gels, similar inductive responses to glucagon or Bt2‐cAMP were observed. Induction by glucagon was shown to be dose‐dependent; a significant response was evident at 0.1 nM gucagon.

Addition of epinephrine to cultured cells at concentration between 10 nM to 0.1 mM was without effect on phosphoenolpyruvate carboxykinase. Induction by Bt2‐cAMP was completely prevented by the simultaneous addition of actinomycin D (0.2 μg/ml) or cordycepin (10 μg/ml) suggesting that the nucletide acts or transcription to increase phosphoenolpyruvate carboxykinase synthesis.
Identification of Some Metabolic Products of 5′‐Deoxy‐5′‐S‐Isobutylthioadenosine, an Inhibitor of Virus‐Induced Cell Transformation
Authors: LAWRENCE Franqoise , RICHOU Mireille , VEDEL Michde , FARRUGIA Giselle , BLANCHARD Pierre , ROBERT‐GERO Malka
Published: March 2005

5′‐Deoxy‐5′‐S‐isobutylthiodenosine (iBuS)5′ Ado has been shown to be rapidly degraded to 5‐deoxy‐5‐S‐isobutylthioribose and adenine in procaryotes. In chick embryo fibroblasts there are two metabolic pathways for (iBuS)5′ Ado degradation: (a) oxidative deamination into 5′‐deoxy‐5′‐S‐isobutylthioinosine (the main product) and (b) hydrolysis into 5‐deoxy‐5‐S‐isobutylthioribose plus adenine. The latter reaction is not due to bacterial contamination, since the same results were obtained under sterile conditions and in chick embryo fibroblasts in culture.

The inhibition of the virus‐induced cell transformation reported by us previously was due to (iBuS)5′ Ado rather than to the main metabolic product of this molecule in chick embryo fibroblasts.
Distribution of AMP‐Deaminase Isozymes in Rat Tissues
Authors: OGASAWARA Nobuaki , GOTO Haruko , YAMADA Yasukazu , WATANABE Tomomasa
Published: March 2005

The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique empolying specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kindney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B–C hybrids.

Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney ad lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throught development. In brain a shift ofB to five isozymes is observed during de3velopment. Isozyme A is the predominant form in muscle throught postnatal development.

AMP deaminase in the regenerating liver was analyzed, but the data indicated that thre was no change of isozyme distribution during hepatic regeneration.
360‐MHz 1H Nuclear‐Magnetic‐Resonance Spectroscopy of Sialyl‐Oligosaccharides form Patients with Sialidosis (Mucolopidosis I and II)
Authors: DORLAND Lambertus , HAVERKAMP Johan , VLIEGENTHART Johannes F. G. , STRECKER Gérard , MICHALSKI Jean‐Claude , FOURNET Bernard , SPIK Geneviève , MONTREUIL Jean
Published: March 2005

360‐MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl‐oligosaccharides isolated form urine of sialidosis patients. Their structures are related to the complex aspareagine‐linked glydan chains of glycoproteins. By correlation of these spectra and comparison with spectra of reference glycopeptides and sialy‐lactose isomers it was possible to assign all signals belinging to anomeric, mannose H‐2, sialic acid H‐3 and N‐acetyl protons. The number of the constituting monosaccharide residues of the oligomers can be obtained by integration of the above‐mentioned singnals. The chemical shifts of the anometic and mannose H‐2 proteins give information about the type of glydan structure (mono‐, bi‐, triantennary) and the presence of terminal sialic acid at eachof the antennas. The chemical shfts of sialic acid H‐3 protons are typical for sialic acid residues in 2→3 or 2→6 linkage ot galactose.
The Synthesis of a Photoreactive Puromycin Analogue and Its Application for Labeling Proteins in the 50‐S Subunit of Escherichia coli Ribosomes
Authors: KRASSNIGG Franz , ERDMANN Volker A. , FASOLD Hugo
Published: March 2005

A photoreactive puromycin analogue, 6‐dimethylamino‐9‐[3‐(p‐azido‐l‐β‐phenlalanylamino)‐3‐deoxy‐β‐ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly(U)‐directed phenylalanine‐incorporation system and by decomposition of isolated polysoomes from Escherichia coli.

The 3H‐labeled puromycin analogue was covalently attache to the 50‐S subunit of isolate 70‐S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bounmd to the protein fraction. The 70‐S proteins were separted by two‐dimensional gel electrophoresis. The proteins labeled primarily were those of the 50‐S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affnity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50‐S particle and probably bellong to the region of the ribosomal A site.
Limited Tryptic Digestion of Human Serum Low‐Density Lipoprotein: Islation and Characterisation of the‐Deficient Particle and of Its Apoprotien
Authors: CHAPMAN M. John , GOLDSTEIN Sonia , MILLS Gervase L. , LAGRANGE D. , TAYLAUR C. E.
Published: March 2005

Limited tryptic digestion of human serum low‐density (LD) lipoprotein (ϱ 1.024–1.045 g/m) under defined conditons permitted isolation by gel filtration chromatography of a stable, protein‐deficient lipoprotein; the liberated protein was separated as a mixture of peptedes of low molecular weight (< 5000). Comparison of the chemical, physical and immunological characterstics of the trypsin‐trated LD‐lipoprotein with those of the native preparation revealed several differences, including (a) a diminished protein content (loss of some 20–25% of the total protien of LD‐lipoprotein) and increased proportions of the various components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD‐lipoprotein (d) an increased net negative charge; and (e) a partial immunological idensity between LD‐lipoprotein and the corresponding trysin‐treated fraction.

While the amino acid compositions of the protein moieties of LD‐lipoprotein and of trypsin‐treated LD‐lipoprotein were essentially identical, trypsin‐treated apo‐LD‐lipoprotein was distinct in its complete solubility in urea‐containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents.

Comparison of the apoproteins of the native and trypsin‐treated LD‐lipoproteins by electrophoretic techniques based on molecular weight revealed a transformation of the high‐molecualar weight material (> 250000) characterstic of apo‐LD lipoprotein into several polypeptide species (10 major forms) ranging in size form 161500 to about 10000. The largest of these (band b1: 161500) could be completely dissociated into smaller components (b2: 93500 and b3: 77000) upon extensive heat treatement at 90°C. Elecstrophoresis of the soluble fraction of apo‐LD‐lipoprotien and of that from its trysin‐treated counterpart in polyacylamide gels containing urea at basic pH showed the didapperance of the small amounts (< 5%) of C apoproteins of apo‐LD‐lipoprotein upon tryptic treatment. These results, which were hightly reproducible in LD‐lipoprotein prepartions from different individuals, suggest that trypsin‐treated LD‐lipoprotein may provide a model for investigation of the organisation and structural role of the principal apoprotein (apoprotein‐B) in the LD‐llipoprotein molecule.
Influence of Magnesium on the Steady‐State‐Derived Order of Substrate Addition and Product Release in tRNA Trp Aminoacylation by Beef Pancreas Tryptophan: tRNA Ligase
Authors: MÉRAULT Guy , GRAVES Pierre‐Vincent , LABOUESSE Bernard , LABOUESSE Julie
Published: March 2005

The order of the partial reactions in the aminoacylation of tRNATrpbeef by tryptophan : tRNA ligase from beef pancreas has been explored by measurements of intial velocity and product inhibition according to the analytical framework described by Cleland [Biochim. Biophys. Acta. 67, 104– 137, 173– 187, 188– 196 (1963)]. The kinetic patterns are suggestive of a ping‐pong mechanism (bi‐uni uni‐bi). The oreder of addition of tryptophan and ATP that one can derive is crucially dependent on the conditions of magnesium concentration used. Random fixation of tryptophan and ATP is observed in studies performed at catalytically optimal magnesium concentration. Linear‐ordered fixation of the two substrates, ATP binding first, is observed under conditions where magnesium concentration is in excess, which are not catalytically optimal. The analysis of the inhibition patterns taking into account the calculated concentrations of teh main active species Mg · ATP2− and Mg · P2O2−7 favors the random substrate‐binding mechanism derived under optimal magnesium conditions. Under both conditions, adjusted and fixed magnesium, the release of products, AMP and tryptophanyl‐tRNATrpbeef is, ordered, AMP released first.

The change in the product nhibition patterns of aminoacylation of tRNATrpbeef by beef pancreas tryptophan: tRNA ligase under different magnesium concentration conditions casts some doubt on the significance of the comparison of mechanisms derived for amino‐acid: tRNA ligases of different sources.
Identification of the Functional Ionic Groups of Papain by pH/Rate Profile Analysis
Authors: ALLEN Kenneth G. D. , STEWART James A. , JOHNSON Phyllis E. , WETTLAUFER David G.
Published: March 2005

The pH dependence of papain catalysis was analyxzed by a scheme which evaluates the kinetic contribution of boith protonated and unprotonated species of fuctional groups inbvolved in catalysi. Kinetic mesurements were made at constant pH, without buffers, by automatic titration. The rate‐determining step for papain‐catalyzed hydrolysis of α‐N‐arginine ethyl ester, determined by nucleophile competition, chantged from acylation below pH 6.5 to mixed acylationdecylation above pH 6.5 kinetic analysis indicated that three prototropic groups governed the pH‐specificity of α‐N‐benozolyl‐l‐arginine ethyl ester hydrolysis. These prototropic groups had pKa values of 4.8, 6.5 to 6.7, and 8.7. Theoretical treatment of the kinetics provided an excellent fit with the experimentally found profile when the contribution of all three prototropic groups was considered. Analysis showed that, in acid, the pathways of paain catalysis werfe functional with either two or three active‐site protons. In base, a single funtional ionic pathway is associated with an active site4 with only one proton. Pathways involving an unprotonated active site are catalytically inperative in both acid and bnase. These results indicate that papin exhibits several catalytically functional ionic pathways. The results are discussed in term of pKa assignments, and the mechanism of papain catalysis.
Adenosine 3′:5′‐Monophosphate and Guanosine 3′:5′‐Monophosphate: Levels and Cyclase Activities in Liver and Adipose Tissue from Diabetic Mice (db/db)
Authors: LEVILLIERS Jacqueline , PAIRAULT Jacques , LECOT Françoise , TOURNEMOLLE Annie , LAUDAT Marie‐Hélène
Published: March 2005

The adenylate and guanylate cyclase activities and the adenosine and guanosine 3′:5′‐monophosphate (cyclic AMP and cyclic GMP) contents have been investigated in the liver and adipose tissue of control homozygote (+m/+m), heterozygote (db+/+m) and diabetic homozygote (db+/db+) mice (C57BL/Ks‐J‐db m strain).

The response to (−)‐isoproterenol of the adenylate cyclase system in isolated fat cells, as well as in adipocyte plasma membranes, from diabetic mice (db+/db+), is impaired as compared to their thin littermates (+m/+m) and also to heterozygote (db+/+m) mice. The increase in cyclic AMP formation elicited by (−)‐isoproterenol was 10‐fold and 4‐fold higher in isolated adipocytes and in adipocyte plasma membranes respectively from lean control as compared to diabetic mice. The cyclic AMP content of adipose tissue from diabetic mice (44.0 ± 7.6 fmol per mg of wet weight) was markedly lower than that from control and heterozygote mice (128.3 ± 41.5 and 128.4 ± 34.5 fmol per mg of wet weight, respectively). In contrast the basal adenylate cyclase activities of liver homogenates and the activties stimulated by glucagon, epinephrine and fluoride were similar in the three groups of animals. Similarly, the hepatic cyclic AMP content did not differ in the three genotypes of mice. These findings indicate that disturbances in cyclic AMP metabolism in adipose tissue from diabetic mice might result from a phenomenon secondary to the diabetic syndrome.

The specific activity of guanylate cyclase in liver and adipocytes progressively decreased from lean control (+m/+m) to heterozygote (db+/+m) to homozygote diabetic (db+/db+) mice; e.g. the guanylate cyclase activity in adipocyte plasma membranes was markedly diminished in diabetic mice (18.9 ± 1.9, 12.7 ± 0.9 and 4.7 ± 1.4 pmol cyclic GMP · min−1· (mg protein)−1 for homozygote control, heterozygote and diabetic mice, respectively). A similar situation was noted in respect of the cyclic GMP levels in the liver and adipose tissue of these animals. Thus, the cyclic GMP content in tissues from diabetic mice represented only 13% and 43% of that in the liver and adipose tissue respectively of control mice. These data are consistent with the existence of a dosage effect of the db gene on cyclic GMP metabolism. On the other hand identical maximal guanylate cyclase activities were obtained after preincubation with Triton X‐100 of adipocyte plasma membranes from the three kinds of mice. At least, there was a 19‐fold and 2.5‐fold stimulation of guanylate cyclase activity by sodium azide in liver homogenates from homozygote diabetic mice and homozygote control, respectively; the hypersensitivity to NaN3 of guanylate cyclase activity in liver and adipose tissue could be related to a disturbance in peroxide metabolism in the tissues of the mutant.
Interferon Induction by a 2′‐Modified Double‐Helical RNA, Poly(2′‐azido‐2′‐deoxyinosinic acid) · polycytidylic acid
Authors: DE CLERCQ Erik , TORRENCE Paul F. , STOLLAR B. David , HOBBS John , FUKUI Toshikazu , KAKIUCHI Nobuko , IKEHARA Morio
Published: March 2005

Although the presence of free 2′‐hydroxyl groups in both strands of a double‐stranded RNA complex has been recognized as one of the major requisites for the interferon‐inducing activity of double‐stranded RNAs, we have found a particular analogue of (I)n· (C)n in which the 2′‐hydroxyls of the purine nucleotide strand were replaced by azido groups, (dIn3)n· (C)n, to be highly effective in inducing interferon. Various other 2′‐azido analogues of (I)n· (C)n and (A)n· (U)n, i.e. (dIn3)n· (br5C)n, (I)n· (dCn3)n, (dAn3)n· (U)n, (A)n· (dUn3)n, (dAn3)n· (rT)n and (dAn3)n· (dUn3)n, were inactive as inducers of interferon. In human fibroblast cultures, the interferon‐inducing activity of (dIn3)n· (C)n equalled that of (I)n· (C)n. In other interferon‐induction systems (primary rabbit kidney cells, mouse L‐929 cells, intact rabbits), (dIn3)n· (C)n was less active than (I)n· (C)n. As assessed by both radio‐chemical and biological means, (dIn3)n· (C)n was more susceptible to degradation by pancreatic ribonuclease and human serum nucleases than was (I)n· (C)n. The tm of (dIn3)n· (C)n was 52.5 °C, as compared to 62.5 °C for (I)n· (C)n, both determined in 0.15 M Na+, pH 7.0. Under the same conditions, (dIn3)n· (br5C)n had a tm of 77 °C and (I)n· (br5C)n had a tm of 87 °C. The reactivity of (dIn3)n· (C)n and (dIn3)n· (br5C)n towards antibodies to double‐stranded RNA was evaluated by quantitative complement fixation, counterimmunoelectrophoresis and competitive radioimmunoassay. In these tests, (dIn3)n· (C)n and (dIn3)n· (br5C)n showed an immunoreactivity pattern comparable to that of (I)n· (C)n and (I)n· (br5C)n.
The Conformation of Histone H5
Authors: AVILES Francisco J. , CHAPMAN George E. , KNEALE Geoff G. , CRANE‐ROBINSON Colyn , BRADBURY E. Morton
Published: March 2005

Treatment of chicken erythrocyte histone H5 with trypsin in a high‐ionic‐strength medium results in very rapid initial digestion and the formation of a ‘limiting’ resistant product peptide. Under these solution conditions the H5 molecule is maximally folded by spectroscopic criteria and it is concluded that the resistant peptide, GH5, represents a globular folded region of the molecule whilst the rapidly digested parts are disordered. The peptide GH5 is shown to comprise the sequence 22–100. In support of this conclusion it is shown that whilst intact histone H5 is hydrodynamically far from being a compact globular shape, peptide GH5 is approximately spherical by hydrodynamic and scattering criteria. Further more, peptide GH5 retains all the α‐helical structure of intact H5 (circular dichroism) and appears to also maintain all the tertiary structure (nuclear magnetic resonance). It follows that in solution at high ionic strength, histone H5 consists of three domains: an N‐terminal disordered region 1–21, a compact globular central domain 22–100 and a long disordered C‐terminal chain 101–185. Structural parallels are drawn with the three‐domain structure of the histone H1 molecule.
Self‐Association of Human Spectrin
Authors: UNGEWICKELL Ernst , GRATZER Walter
Published: March 2005

Depending on conditions of extraction from the membrane, spectrin may be recovered either as a dimer or a tetramer. It is demonstrated that these species are linked by a simple equilibrium, and can be readily interconverted. At low temperature, however, either species is kinetically trapped, and no interconversion occurs over periods of up to several days. In the range 25‐40 °C, equilibrium is achieved on a time scale of hours to minutes. First‐order and second‐order rate constants and thermodynamic parameters for the equilibrium have been determined. The large activation energy indicates that conformational effects are rate‐limiting. In the absence of other proteins, no associated states higher than the tetramer are formed. Neither phosphorylation of the spectrin with endogenous kinase, nor dephosphorylation with phosphatase have any effect on the dimer‐tetramer equilibrium, neither do they promote formation of higher associated states. It is therefore improbable that phosphorylation‐dependent shape changes in the erythrocyte are related to the association of spectrin per se, and are likely instead to be the direct result of a spectrin‐actin interaction. The dimer‐tetramer equilibrium is strongly affected by ionic strength, and at low salt concentrations, such as those used to extract spectrin from membranes, the dimer becomes strongly favoured. Thus the recovery of tetramer by extraction at low temperature implies that this is the basic unit present in the membrane. Calcium and magnesium ions cause further association to higher oligomers, though only at high concentrations (in the millimolar range) of the cation.
Isolation and Characterization of the Hemoglobin from the Lanceolate Fluke Dicrocoelium dendriticum
Authors: TUCHSCHMID Peter E. , KUNZ Peter A. , WILSON Kenneth J.
Published: March 2005

The hemoglobin of the flatworm Dicrocoelium dendriticum, a lanceolate fluke which infests the hepatic ducts of certain mammals, has been isolated by gel filtration and ion‐exchange chromatography. The molecular weight of the denatured protein was found to be 15500, a value in the same range as hemoglobin subunits. The fact that the native hemoglobin has an apparent molecular weight of 22000 in 0.01 M phosphate buffer, pH 7.4, suggests limited aggregation. The protein contains, as all other myoglobins and hemoglobins, one molecule of non‐covalently associated ferroprotoporphyrin IX per polypeptide chain. It forms the same ligand derivatives with very similar spectral properties as vertebrate hemoglobins. The high oxygen affinity (p50 is 0.07–0.1 mmHg or 9.3–13.3 Pa at 20 °C and pH 7.0) and the absence of heme‐heme interaction (Hill coefficient nH= 1.0) are properties which this heme protein shares with other monomeric hemoglobins from invertebrate and lower vertebrate organisms.

The native hemoglobin exists in two forms, having isoelectric points of 4.51 and 4.53, which do not differ in their amino‐acid compositions. Dansylation indicated that the amino‐terminal amino‐acid residue is alanine. The carboxy‐terminal sequence, determined by carboxypeptidase A digestion of the globin, is ‐His‐Ala‐Leu.
The Capacity of Polyadenylated RNA from Myogenic Cells Treated with Actinomycin D to Direct Protein Synthesis in a Cell‐Free System
Authors: KESSLER‐ICEKSON Gania , SINGER Robert H. , YAFFE David
Published: March 2005

Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin‐D‐treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell‐free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell‐free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half‐life ranging between 11 and 40 h. No fast‐decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4–6 h following application of actinomycin D, the capacity of RNA to stimulate incorporation of amino acids into total acid‐insoluble material increased by 20–30%. The synthesis of specific products increased by up to 100%. The possibility that the fast‐decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed.
Study of Actin and Its Interactions with Heavy Meromyosin and the Regulatory Proteins by the Pulse Fluorimetry in Polarized Light of a Fluorescent Probe Attached to an Actin Cysteine
Authors: TAWADA Katsuhisa , WAHL Philippe , AUCHET Jean‐Claude
Published: March 2005

The decay of anisotropy of the N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)‐ethylenediamine fluorescence attached to cysteine‐373 of actin can be characterized by two correlation times θ1 and θ2. θ1 has a value of several nanoseconds and is thought to represent some local protein motion. θ2 is of the order of several hundreds of nanoseconds. Its value increases with actin concentration. It represents an average of the G and F actin correlation times. When actin interacts with heavy meromyosin, θ2 increases and becomes infinite at a molar ratio of one heavy meromyosin molecule per four actin protomers. It is concluded that a definite complex is then formed between F actin and heavy meromyosin. In the same time, G actin concentration becomes equal to zero. Finally, when F actin forms a complex with the regulatory proteins tropomyosin and troponin, the value of θ2 is greater in the absence than in the presence of Ca2+. This result indicates that micromolar concentrations of Ca2+ induces a conformation change of the complex of F actin with the regulatory proteins.