bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of Human Hypoxanthine/Guanine Phosphoribosyltransferase
Authors: MUENSCH Helmut , YOSHIDA Akira
Published: March 2005

Human hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) was purified from red blood cells by the following two methods. Method A includes (a) elimination of hemoglobin by DEAE‐cellulose, (b) DEAE‐Sephadex chromatography, (c) specific elution of the enzyme from CM‐Sephadex by pyrophosphate and (d) Sephadex G‐100 gel filtration. Method B includes (a) elimination of hemoglobin by DEAE‐cellulose, (b) acid treatment at pH 4.5, (c) ammonium sulfate fractionation, (d) DEAE‐Sephadex chromatography, (e) heat treatment at 85°C and (f) Sephadex G‐100 gel filtration.

Homogeneous enzyme preparation was obtained by the two methods with 8000–9000‐fold purification. The sedimentation coefficient (s20, w) was 5.5–5.6 S, and the molecular weight of the enzyme was estimated at about 85000 by the sedimentation equilibrium method. The subunit molecular weight of the untreated protein and S‐carboxymethylmaleyl protein was estimated as 41000–45000 by the sedimentation equilibrium method in the presence of guanidine hydrochloride. However, the subunit size estimated by the sodium dodecylsulfate gel electrophoresis was only 26000. Amino acid composition of the enzyme was determined. Glucosamine, sialic acid and hexose were not detected in the enzyme.
Characterization of a Protein Factor Stimulating RNA Synthesis by DNA‐Dependent RNA Polymerase II from Plant Cell Cultures
Authors: LINK Gerhard , RICHTER Gerhard
Published: March 2005

During purification of DNA‐dependent RNA polymerase II (or B) from cell cultures of parsley a protein fraction was separated by phosphocellulose chromatography which enhanced RNA synthesis in the presence of native homologous DNA. This ‘stimulatory factor’ was characterized in respect to some effects on the reaction catalyzed by RNA polymerase II. In the presence of the factor the metal ion requirements as well as the ionic strength for optimal RNA synthesis were markedly changed; addition of the factor to RNA polymerase II purified by cellulose chromatography restored those enzyme properties which had apparently changed upon this purification step. The chain length of the RNA product synthesized is favouring the view that the factor acts mainly by stabilizing the elongation step during transcription.

The stimulatory factor was further purified by several steps of column chromatography. As derived from the results of gel electrophoresis under denaturing conditions the factor consists of several small polypeptides. Those of Mr= 26000, 25000 and 14000 apparently have counterparts among the smaller subunits of highly purified RNA polymerase II from parsley cells. Another polypeptide of the factor, with Mr= 30000, was only found in those preparations of RNA polymerase II which had not been subjected to phosphocellulose chromatography.
Primary Structure of the B‐Chain of Human Plasmin
Authors: WIMAN Björn
Published: March 2005

The primary structure of the human plasmin B‐chain has been determined. It consists of 230 residues divided in three cyanogen bromide fragments: The amino‐terminal 24 residues, the carboxy‐terminal three residues and the middle 203 residues. Sequence determination was performed on the tryptic and the chymotryptic peptides obtained from the main cyanogen bromide fragment of this chain. Owing to similarities between some of the overlapping chymotryptic peptides, two different sequences were possible from these results. However, since the homologies with the pancreatic serine proteases and also the B‐chains of thrombin and factor XA are pronounced, the arrangement still could be settled. By peptic digestion of partially reduced and S‐carboxymethylated B‐chain it was shown that there are two interchain disulphide bridges, which connect the A and B‐chains of plasmin, involving Cys‐5 and Cys‐105 from the B‐chain. The intrachain disulphides in the B‐chain seem to be situated exactly as in chymotrypsin as partly judged from homologies.
Increased de Novo Pyrimidine Nucleotide Synthesis in Liver Induced by Ammonium Ions in Amounts Surpassing the Urea Cycle Capacity
Authors: PAUSCH Jürgen G. , GEROK Wolfgang , KEPPLER Dietrich O. R.
Published: March 2005

An increased rate of de novo pyrimidine nucleotide synthesis is induced in rat liver by administration of ammonium ions. Specific enzymatic analyses demonstrate a marked elevation of the hepatic contents of orotate, UTP, UDP‐glucose, total uracil and cytosine 5′‐nucleotides. Inhibition of uridylate formation by means of 6‐azauridine suppresses the effect of ammonium ions on uracil nucleotide contents and intensifies the accumulation of orotate. After administration of ammonium ions, de novo uridylate synthesis continues at an elevated rate when the hepatic contents of ammonium ions have returned to the normal range. A mediating role of the elevated glutamine levels in the in‐ crease of pyrimidine synthesis induced by ammonium ions has been excluded by an inhibition of glutamine synthetase by methionine sulfoximine.

The following evidence indicates that mitochondrial carbamoyl phosphate synthesis from ammonium ions serves as the source of the excess carbamoyl phosphate channeled into cytoplasmic pyrimidine synthesis and leading to an increased formation of orotate and uridylate: (a) acceleration of mitochondrial carbamoyl phosphate consumption by ornithine in the urea cycle prevents the increase of pyrimidine synthesis induced by ammonium ions; (b) inhibition of ornithine carbamoyltransferase by the ornithine analogs norvaline and 2,4‐diaminobutyrate mimics the effect of excess ammonium ions and leads‐to an increased formation of pyrimidines; (c) ascites hepatoma cells deficient in mitochondrial ammonia‐dependent carbamoyl‐phosphate synthetase are unable to increase their rate of pyrimidine synthesis in the presence of ammonium ions.

Ornithine has no inhibitory effect on the elevated rate of pyrimidine synthesis elicited by UTP deficiency. This indicates that an efflux of mitochondrial carbamoyl phosphate plays a negligible role in the absence of excess ammonium ions in intact liver. Surplus ammonium ions, however, lead to a state of increased de novo pyrimidine nucleotide synthesis in which the feedback regulation of this pathway by UTP is bypassed.
A Phosphorus‐Magnetic‐Resonance Study of the Interaction of Mg2+ with Adenyl‐5′‐yl Imidodiphosphate
Authors: TRAN‐DINH Son , ROUX Madeleine
Published: March 2005

The interaction of Mg2+ ions with adenyl‐5′‐yl imidodiphosphate, AMP‐P(NH)P, has been studied at basic and acidic pH values by phosphorus magnetic resonance spectroscopy in aqueous solution. The results suggest that Mg2+ binds simultaneously to one (or both) of the two free oxygen atoms of the β‐phosphate moiety and to the nitrogen atom of the phosphate chain (Pα‐O‐Pβ‐N‐Pγ). The interaction arises from 1:1 complexing of Mg2+ to AMP‐P(NH)P. The mode of the Mg2+ binding on the phosphate chain remains the same at both basic and acidic pH values. As in the case of ATP and ADP, the association of Mg2+ reduces the pK by about 1.5 units. On the other hand phosphorus titration curves showed that when the phosphate chain does not possess the regular periodicity (O‐Pα‐O‐Pβ‐X‐Pγ‐O,X ≠ O) as in the case of ATP, protonation of the terminal phosphate group may induce a 31P chemical shift variation less important for this group than for the preceding one.
A Comparative Analysis of the Protein Components of Plasma Membranes Isolated from Differentiated and Undifferentiated Mouse Neuroblastoma Cells in Tissue Culture
Authors: GARVICAN John H. , BROWN Geoffrey L.
Published: March 2005

The plasma membranes of the cells of mouse neuroblastoma clone NB41A, were isolated without fixation by hardening procedures and were characterised by their enzyme activities and by their morphology in the light and electron microscopes. The membranes were prepared from two kinds of differentiated monolayer cultures; one in which the cells were induced to form long axon‐like processes by the addition of N6,O2′‐dibutyryladenosine 3′:5′‐monophosphate to the culture medium, and the other in which the cells were induced to form processes by the addition of 5‐bromo‐2′‐deoxyuridine. The proteins from the solubilised membranes were compared with similar preparations from the membranes of undifferentiated cells, grown in suspension, by sodium dodecylsulphate polyacrylamide gel electrophoresis, using the incorporation of radioactive amino acids and l‐fucose into proteins in the cultures to follow the differences in the patterns of polypeptide synthesis and glycosylation of the membrane proteins.

The differentiated cells induced with either inducer show an increased incorporation of l‐fucose into two distinct components with molecular weights of 60000 and 70000. The two types of induced cells differ from each other in that N6,O2′‐dibutyryladenosine 3′:5′‐monophosphate stimulates both glycosylation and protein synthesis, with a relative increase in the low molecular weight proteins, but 5‐bromo‐2′‐deoxyuridine stimulates mostly increased glycosylation of the membrane proteins.
Involvement of the cytoplasmic membrane in nitrogen fixation by azotobacter vinelandii
Authors: HAAKER Huub , VEEGER Cees
Published: March 2005

1. Azotobacter vinelandii cells were ruptured by using a French pressure cell, by lysozyme treatment, or by osmotic shock. The rates of sedimentation of nitrogenase from these extracts were compared and related to the rates of sedimentation of marker enzymes. No evidence was found for the so‐called particulate nitrogenase or an interaction between nitrogenase and cytoplasmic membrane vesicles. Nitrogenase sediments as a complex and the rate of sedimentation is comparable with that of pyruvate dehydrogenase complex.

2. The oxygen stability of nitrogenase from Azotobacter is not caused by the presence of cytoplasmic membranes in extracts but by a complexation of the nitrogenase components with an Fe‐S protein. Recombination experiments showed that maximum stabilization against oxygen was only obtained at certain ratios of the Fe‐S‐protein and nitrogenase complex. It is proposed that the switch‐off state in Azotobacter cells is caused by the oxidation of flavodoxin hydroquinone rather than by a reversible inactivation of the nitrogenase.

3. A membrane‐bound NAD(P)H‐flavodoxin oxidoreductase was detected. Evidence was obtained that flavodoxin might also be membrane‐bound.

4. A proposal is given for electron donation to nitrogenase in Azotobacter. In this proposal the generation of reducing equivalents for nitrogenase is localized in the cytoplasmic membrane and mediated by the NADH‐flavodoxin oxidoreductase. The pH gradient which is generated by membrane energization, is used to reduce flavodoxin to its hydroquinone form in order to achieve a redox potential low enough to reduce nitrogenase.
Adenosine Diphosphoribosylation of Certain Basic Chromosomal Proteins in Isolated Trout Testis Nuclei
Authors: WONG Norman C. W. , POIRIER Guy G. , DIXON Gordon H.
Published: March 2005

Isolated trout testis nuclei rapidly incorporate [α‐32P]NAD+into chromosomal proteins. Three proteins, very‐lysine‐rich histone (H1), a specific trout chromosomal protein (H6) and the sperm‐specific protamines, incorporate the label as adenosine diphosphoribosyl (ADP‐Rib) residues. No significant labeling of the nucleosomal ‘core’ histones H2A, H2B, H3 and H4 was observed. The linkage of the [32P](ADP‐Rib) residues to each protein was very labile at pH values < 7.0 but by working at acidic pH and low temperatures the ADP‐Rib label could be shown to be covalently bound to protein by gel electrophoresis and ion‐exchange chromatography. The [32P]ADP‐Rib chains could be removed by digestion with snake venom phosphodiesterase with the formation of AMP and phosphoribosyl‐AMP.
Purification of Aldolase C from Rat Brain and Hepatoma
Authors: HATZFELD Antoinette , ELION Jacques , MENNECIER Francois , SCHAPIRA Fanny
Published: March 2005

An isolation procedure for rat brain aldolase C has been developed which also permits the isolation of aldolase C from experimental hepatomas.

Certain enzymatic properties (specific activity and Michaelis constant towards the two specific substrates: fructose 1,6‐bisphosphate and fructose 1‐phosphate) and physico‐chemical properties (molecular weight, N‐terminal amino‐acid) of the two enzymes have been studied and compared. Moreover, an amino‐acid analysis has been carried out for rat brain aldolase C.

Within experimental errors, the two enzymes appear to be identical.
Dimerization of Papain Induced by Mercuric Chloride and a Bifunctional Organic Mercurial
Authors: SLUYTERMAN Lamoraal A. AE. , WIJDENES Jacob , VOORN Gysbertus
Published: March 2005

The bifunctional mercurial meso‐1,4‐bis(acetatomercuri)‐2,3‐diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium.

The conformational change of papain necessary for this reaction is discussed.
Reaction of Human Choriomammotropin with Hydrogen Peroxide
Authors: HOUGHTEN Richard A. , HAO LI Choh
Published: March 2005

Treatment of human choriomammotropin with hydrogen peroxide in the absence of denaturant was found to oxidize five of its six methionine residues to sulfoxide; the residue not oxidized was found to be methionine‐170. This derivative was identical to the native hormone by exclusion chromatography, circular dichroism spectra, and rate of tryptic digestion, but suffered a very substantial drop in biological activity. In the presence of 8 M urea all of the methionines could be oxidized with hydrogen peroxide or alkylated with iodoacetic acid. The physical properties of these completely methionine‐modified derivatives were significantly changed and biological activity had again been very substantially decreased. In control experiments, treatment of the native hormone with 8 M urea did not affect its physical, spectral or biological properties. Amino acid analysis of the derivatives was completely in accord with that expected.
Antigenicity of Bovine Ribonuclease Modified at Tyrosine or Arginine Residues
Authors: VAN DER ZEE Ruurd , JAN DUISTERWINKEL Feitse , WELLING Gjalt W.
Published: March 2005

Bovine pancreatic ribonuclease was treated with tetranitromethane at a molar excess of 100 mol/mol protein at pH 8.0.

Spectral studies showed that the nitration reaction can be better quantitated from the absorbance at 381 nm than from the generally used absorbance at 428 nm.

With analytical isoelectric focusing it was found that the reaction product contained various derivatives, nitrated to a different extent.

The two derivatives with the highest pI value were isolated by preparative isoelectric focusing in layers of granulated gel. Following performic acid oxidation, modified ribonuclease was digested successively with chymotrypsin and thermolysin. Amino acid analyses of the nitrated peptides showed that tyrosine‐115 was nitrated in the component with the highest pI value, while tyrosines‐76 and 115 were modified in the other nitrated component. The latter derivative exhibited the same enzymatic activity and antigenicity as unmodified ribonuclease.

Bovine pancreatic ribonuclease was also modified with 1,2‐cyclohexanedione. After ion‐exchange chromatography and tryptic digestion of the oxidized protein it was determined that arginine residues 39 and 85 were modified for more than 90%, while arginine residue 10 was modified for 30%. Arginine residue 33 remained unchanged. The enzymatic activity of the cyclohexanedione‐treated ribonuclease was less than that of the unmodified protein and there was no loss of antigenic reactivity.

It was concluded that tyrosine residues 76 and 115, and arginine residues 39 and 85 do not belong to antigenically relevant regions of bovine pancreatic ribonuclease.
Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences
Authors: SIPPEL Albrecht E. , GRONER Bernd , HYNES Nancy , SCHÜTZ Günther
Published: March 2005

Total rat liver poly(A)‐containing polysomal mRNA was size‐fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8–14‐S mRNA fraction and separated into sequences representing abundant and non‐abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that:

1. All abundant 8–14‐S poly(A)‐containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length.

2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18‐S RNA and 2% are between 37 S and 44 S.

3. The majority of small messenger‐containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8–14‐S mRNA.
The Role of Cytochrome P‐450 in the Hydroperoxide‐Catalyzed Oxidation of Alcohols by Rat‐Liver Microsomes
Authors: RAHIMTULA Anver D. , O'BRIEN Peter J.
Published: March 2005

The organic hydroperoxide cumene hydroperoxide is capable of oxidizing ethanol to acetaldehyde in the presence of either catalase, purified cytochrome P‐450 or rat liver microsomes. Other hemoproteins like horseradish peroxidase, cytochrome c or hemoglobin were ineffective. In addition to ethanol, higher alcohols like 1‐propanol, 1‐butanol and 1‐pentanol are also oxidized to their corresponding aldehydes to a lesser extent. Other organic hydroxyperoxides will replace cumene hydroperoxide in oxidizing ethanol but less effectively. The cumene‐hydroperoxide‐dependent ethanol oxidation in microsomes was inhibited partially by cytochrome P‐450 inhibitors but was unaffected by catalase inhibitors. Phenobarbital pretreatment of rats increased the specific activity of the cumene‐hydroperoxide‐dependent ethanol oxidation per mg of microsomes about seven‐fold. The evidence suggests that cytochrome P‐450 rather than catalase is the enzyme responsible for hydroperoxide‐dependent ethanol oxidation. However, when H2O2 is used in place of cumene hydroperoxide, the microsomal ethanol oxidation closely resembles the catalase system.
Ca2+‐Dependent Allosteric Regulation of Nicotinamide Nucleotide Transhydrogenase from Pseudomonas aeruginosa
Authors: HÖJEBERG Bo , RYDSTRÖM Jan
Published: March 2005

1. The addition of Ca2+ to purified nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa results in a 2–8‐fold increase in flavin fluorescence, closely resembling that induced by 2′‐AMP (adenosine 2′‐monophosphate). The response to Ca2+ is sigmoidal, with a Hill‐coefficient of 2.6. Reduction of thionicotinamide adenine dinucleotide (oxidised form) by NADH responds in a similar fashion to Ca2+ and 2′‐AMP.

2. Mg2+ inhibits the Ca2+‐dependent activation in a competitive fashion, whereas no effect of Mg2+ is observed when the enzyme is activated with 2′‐AMP.

3. The pH dependencies of the activations by 2′‐AMP and Ca2+ are different. The activation by Ca2+ shows little or no dependence on pH, whereas higher concentrations of 2′‐AMP are required to obtain the same activation when the pH is increased.

4. The addition of both Ca2+ and 2′‐AMP causes a dramatic decrease in the concentration of Ca2+ required for half‐maximal stimulation.

5. A negative cooperative effect of NADP+ has been demonstrated indirectly, i.e. addition of NADP+ increases the concentration of Ca2+ required for half‐maximal stimulation.

6. These results are interpreted as indicating the existence of separate allosteric binding sites for Ca2+ and 2′‐AMP. Furthermore, Ca2+ is proposed to act as a heterotropic ligand. The results are discussed in terms of current allosteric models.
Thyroglobulin Messenger Ribonucleic Acid Translation in vitro
Authors: CHEBATH Judith , CHABAUD Odile , CARTOUZOU Guy , LISSITZKY Serge , BEČAREVIČ Aleksander
Published: March 2005

Total polysomes were obtained by homogenization of sheep thyroid glands in Tris‐Cl‐buffered sucrose, pH 7.5, treatment with 2% Triton X‐100 and centrifugation of the magnesium‐treated 27000 ×g supernatant through 1 M sucrose. Purification of thyroglobulin‐specific polysomes was performed by indirect immunoprecipitation with rabbit antibodies to sheep thyroglobulin and donkey antibodies to rabbit immunoglobulin G. Thyroglobulin polysomes represented 30% of total polysomes. Synthesis of thyroglobulin peptides in a reticulocyte cell‐free system, programmed with total RNA from immunoprecipitated polysomes, was 3 times higher than when total RNA from unfractionated polysomes was used. The size of the poly(A)‐containing thyroglobulin mRNA was estimated by the capacity of RNA fractions obtained by sucrose density gradient centrifugation of total or immunopurified polysomal RNA to hybridize with 3H‐labeled poly(U) and to stimulate thyroglobulin peptide synthesis in a reticulocyte lysate. 65% of the thyroglobulin messenger activity was located in a 33–36‐S RNA peak and 35% in zones corresponding to 30‐S and 26‐S RNA species.

The thyroglobulin‐specific immunoprecipitable peptides synthesized in the reticulocyte lysate from the 33–36‐S RNA were shown by polyacrylamide gel electrophoresis in denaturing conditions to contain a major component co‐migrating with the constituent peptide chain of sheep thyroglobulin (Mr 330000). Other components migrated with Mr of about 210000 and 120000.
The Complete Amino‐Acid Sequence of Histone H2B(1) from Sperm of the Sea Urchin Parechinus angulosus
Authors: STRICKLAND Marie , STRICKLAND Walter N. , BRANDT Wolf F. , VON HOLT Claus
Published: March 2005

Two histones have been isolated and purified from sperm cells of the sea urchin Parechinus angulosus. They have been designated sperm histone H2B(1) Parechinus and sperm histone H2B(2) Parechinus on criteria of solubility and partial sequence homology with H2Bcalf.

H2B(1) consists of a polypeptide chain of the following 144 amino acid residues: Pro‐Ser‐Gln‐Lys‐Ser‐Pro‐Thr‐Lys‐Arg‐Ser‐Pro‐Thr‐Lys‐Arg‐Ser‐Pro‐Thr‐Lys‐Arg‐Ser‐Pro‐Gln‐Lys‐Gly‐Gly‐Lys‐Gly‐Gly‐Lys‐Gly‐Ala‐Lys‐Arg‐Gly‐Gly‐Lys‐Ala‐Gly‐Lys‐Arg‐Arg‐Arg‐Gly‐Val‐Gln‐Val‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Glu‐Ser‐Tyr‐Gly‐Ile‐Tyr‐Ile‐Tyr‐Lys‐Val‐Leu‐Lys‐Gln‐Val‐His‐Pro‐Asp‐Thr‐Gly‐Ile‐Ser‐Ser‐Arg‐Ala‐Met‐Ser‐Val‐Met‐Asn‐Ser‐Phe‐Val‐Asn‐Asp‐Val‐Phe‐Glu‐Arg‐Ile‐Ala‐Ala‐Glu‐Ala‐Gly‐Arg‐Leu‐Thr‐Thr‐Tyr‐Asn‐Arg‐Arg‐Ser‐Thr‐Val‐Ser‐Ser‐Arg‐Glu‐Val‐Gln‐Thr‐Ala‐Val‐Arg‐Leu‐Leu‐Leu‐Pro‐Gly‐Glu‐Leu‐Ala‐Lys‐His‐Ala‐Val‐Ser‐Glu‐Gly‐Thr‐Lys‐Ala‐Val‐Thr‐Lys‐Tyr‐Thr‐Thr‐Ser‐Arg. In a previously published partial sequence serine was incorrectly assigned to position 30.

A comparison of the carboxyl‐terminal two thirds of H2B(1) Parechinus and H2Bcalf reveals that 79% of the corresponding positions are occupied by identical amino acid residues. Much of the variability occurs in a short polar region of 20 amino acid residues. The remainder of C‐terminal regions is hydrophobic and almost completely invariable.

A comparison of the amino‐terminal one third of H2B(1) Parechinus and H2Bcalf reveals that they are both highly basic with a considerable homology of the position of basic amino acids. Further, H2B(1) Parechinus possesses an amino terminal extension of 19 amino acids. These 19 amino acids can be recognized as a repeating pentapeptide.
The Complete Amino‐Acid Sequence of Histone H2B(2) from Sperm of the Sea Urchin Parechinus angulosus
Authors: STRICKLAND Walter N. , STRICKLAND Marie , BRANDT Wolf F. , VON HOLT Claus
Published: March 2005

Sperm histone H2B(2)Parechinus consists of a single polypeptide chain of the following 143 amino acids: Pro‐Arg‐Ser‐Pro‐Ala‐Lys‐Thr‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Ser‐Arg‐Lys‐Ala‐Ser‐Pro‐Lys‐Arg‐Gly‐Gly‐Lys‐Gly‐Ala‐Lys‐Arg‐Ala‐Gly‐Lys‐Gly‐Gly‐Arg‐Arg‐Arg‐Arg‐Val‐Val‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Glu‐Ser‐Tyr‐Gly‐Ile‐Tyr‐Ile‐Tyr‐Lys‐Val‐Leu‐Lys‐Gln‐Val‐His‐Pro‐Asp‐Thr‐Gly‐Ile‐Ser‐Ser‐Arg‐Ala‐Met‐Ser‐Val‐Met‐Asn‐Ser‐Phe‐Val‐Asn‐Asp‐Val‐Phe‐Glu‐Arg‐Ile‐Ala‐Gly‐Glu‐Ala‐Ser‐Arg‐Leu‐Thr‐Ser‐Ala‐Asn‐Arg‐Arg‐Ser‐Thr‐Val‐Ser‐Ser‐Arg‐Glu‐Ile‐Gln‐Thr‐Ala‐Val‐Arg‐Leu‐Leu‐Leu‐Pro‐Gly‐Glu‐Leu‐Ala‐Lys‐His‐Ala‐Val‐Ser‐Glu‐Gly‐Thr‐Lys‐Ala‐Val‐Thr‐Lys‐Tyr‐Thr‐Thr‐Ser‐Arg. In a previously published partial sequence positions 25, 34, 40 and 41 were incorrectly assigned.

The amino‐terminal one third of sperm histone H2B(2)Parechinus is highly basic containing a repeating pentapeptide occurring over the first 24 residues. This repeating pentapeptide is different to that of sperm histone H2B(1)Parechinus. In this region there is very little amino acid sequence homology between H2B(1)Parechinus, H2B(2)Parechinus on the one side and H2Bcalf thymus on the other side, but all three proteins are highly basic in their N‐terminal parts.

The carboxyl‐terminal two thirds of sperm histone H2B(2) is hydrophobic except for a short polar region of 20 amino acids. The carboxyl‐terminal region shows a very high amino acid sequence homology between the three H2B histones. Much of the variability occurs in the short polar region whereas the hydrophobic regions are almost completely constant. The majority of the amino acid substitutions that occur in the carboxyl end are conservative.

The three H2B protein sequences are compared and the functional and evolutionary significance of the changes is discussed.
Characterization of the Protein Moiety of Messenger Ribonucleoprotein Complexes from Duck Reticulocytes by Two‐Dimensional Polyacrylamide Gel Electrophoresis
Authors: MUELLER Rita U. , CHOW Virginia , GANDER Eugen S.
Published: March 2005

The protein moiety of duck globin messenger ribonucleoprotein complexes isolated by oligo(dT)‐cellulose chromatography or by sucrose gradient centrifugation was analysed by two‐dimensional polyacrylamide gel electrophoresis under conditions where the separation in the first dimension occurs according to charge and in the second according to molecular weight.

By comparing the pattern of protein from the mRNA · protein complex with that of ribosomal subunits we found that two acidic proteins with an identical molecular weight of about 49000 and three basic proteins of about Mr 56000, 64000 and 73000 were associated with the duck globin mRNA but were absent from either puromycin/high‐salt‐derived or ‘run‐off’ ribosomal subunits.

The comparison of the proteins from the complex with mRNA with those found in the 0.5 M KCl wash, commonly used as the source of initiation factors, showed also that only the 49000‐Mr protein from the complex could possibly be present in the 0.5 M KCl wash of polyribosomes; proteins with mobilities similar to the other three proteins complexed with mRNA were not detected in the salt wash of polyribosomes.
Immunogenic Contaminants in Mouse Nerve‐Growth Factor
Authors: CARSTAIRS Jill R. , PEARCE Frederick L. , VERNON Charles A. , WALTER Stephen J. , EDWARDS D. Caird
Published: March 2005

Experiments are described that confirm the presence of γ‐globulin in standard preparations of mouse nerve growth factor (7‐S complex and β subunit) and show that antibodies to this protein are present in horse antisera to the growth factor. These antibodies may be partially removed by absorption with soluble antigen and totally removed by affinity chromatography on columns of insolubilised antigen. These procedures do not affect the potency of the antiserum in vitro or in vivo. Contaminating globulin may be removed from samples of the isolated β subunit by gel filtration to give an immunochemically pure preparation which will permit more meaningful studies on nerve growth factor and its antiserum.
The Disialosyl Group of Glycoproteins
Authors: FINNE Jukka , KRUSIUS Tom , RAUVALA Heikki , HEMMINKI Kari
Published: March 2005

A sensitive method for the determination of disialosyl (α‐N‐acetylneuraminyl‐(2‐8)‐N‐acetyl‐neuraminyl) groups was developed. The method is based on the quantitation of the amount of 8‐O‐substituted neuraminic acid by methylation analysis using mass fragmentographic detection with two different m/e values. Lability of the 8‐O‐substituent of neuraminic acid to neuraminidase is taken as an indication for the presence of a disialosyl sequence.

Considerable differences in the amount of disialosyl groups were observed between glycopeptides from different tissues of the rat. The proportion of 8‐O‐substituted neuraminic acid as compared to total glycopeptide‐bound neuraminic acid was 8.5% in brain, 3.1% in liver, 2.6% in diaphragmatic muscle and 3.0% in small intestinal mucosa, whereas glycopeptides derived from kidney, gastric mucosa, erythrocytes and plasma did not contain disialosyl groups.

The disialosyl groups were enriched in glycoproteins of the membranous fractions of liver and brain, whereas only small amounts were observed in the soluble fractions. The proportion of glycopeptide‐bound 8‐O‐substituted neuraminic acid in the brain of young rats (13.9%) was higher than in the brain of adult animals. The highest proportion of 8‐O‐substituted neuraminic acid (19.8%) was observed in glycopeptides of the plasma membrane fraction of the young rat brain.
Studies on Hemoglobin Tryptophany1 Contact Residues in the Haptoglobin · Hemoglobin Complex
Authors: ROGARD Monique , WAKS Marcel
Published: March 2005

Hemoglobin and apohemoglobin bind haptoglobin in the same molar ratio. Structural studies on haptoglobin · hemoglobin complex do not suggest any important structural changes in either protein upon binding. However, when apohemoglobin is bound to haptoglobin, a marked reduction in secondary structure, attributed to unfolding of globin chains, has been observed.

Here we describe some properties of the haptoglobin · apohemoglobin (Hp · apoHb) complex, prepared by isoelectric focusing in the presence of an excess of haptoglobin. This complex does not exhibit the irreversibility of complexes obtained with hemoglobin in identical experimental conditions.

The ‘freezing’ of the conformation of apohemoglobin upon binding to haptoglobin has been studied by fluorescence quenching experiments carried out in the presence of 8 M acrylamide. Changes in conformation of haptoglobin upon binding to apohemoglobin have been detected by titration of the exposed tryptophans using N‐bromosuccinimide.

Comparison of the additivity of exposed tryptophans in the complexes reveal that two tryptophans become inaccessible in the complex formation of haptoglobin with hemoglobin but not with apohemoglobin. These tryptophans, probably located on the α1β2 contact interface of hemoglobin, have been tentatively identified as Trp‐C3(37)β.
α‐Galactosidase from Saccharomyces carlsbergensis
Authors: LAZO Pedro S. , OCHOA Amparo G. , GASCÓN Santiago
Published: March 2005

1. The α‐galactosidase of Saccharomyces carlsbergensis is an inducible enzyme which is localized mainly outside the cell membrane and which is secreted into the culture medium in increasing amounts during the growth cycle.

2. The soluble form of α‐galactosidase localized inside the cell appears to have the same characteristics as the external one, contrasting with the different forms found in the case of invertase. Although some activity is membrane‐bound, this activity, when solubilized with detergent, has the same characteristics as the external form of the enzyme.

3. A procedure has been developed by which the enzyme has been purified using batch adsorption with DEAE‐Sephadex and column chromatography in DEAE‐Sephadex, DEAE‐cellulose and Sephadex G‐200, using the supernatant of a culture of Saccharomyces carlsbergensis grown in yeast nitrogen base complemented with galactose.

4. The purified enzyme, which is homogeneous by chromatographic criteria and polyacrylamide gel electrophoresis, appears to be glycoprotein.

5. Invertase copurifies with the α‐galactosidase but because of its lower stability, together with the fact that the synthesis of both enzymes can be controlled separately, it was possible to obtain preparations in which the contaminant activity was approximately 1%.
Drug Oxidations Dependent on Cytochrome P‐450 in Isolated Hepatocytes
Authors: WEIGL Klaus , SIES Helmut
Published: March 2005

1. Isolated hepatocytes from male rats pretreated with phenobarbital were used to study metabolic effects associated with monooxygenase activity dependent on cytochrome P‐450. The hepatocytes metabolized an added drug substrate, [dimethylamino‐14C]aminopyrine (1 mM) at an N‐demethylation rate of 1.31 ± 0.1 μmol × min−1× g (dry weight of hepatocytes)−1. The overall ATP/ADP ratios were 5.0 in the presence of aminopyrine, similar to the controls.

2. The levels of citrate and isocitrate decreased, and the citrate/2‐oxoglutarate ratio decreased to 55% of the control in the presence of drug substrates (aminopyrine, ethylmorphine, hexobarbital), correlating well with the decrease in the overall NADPH/NADP+ ratio observed earlier with the perfused liver. It is concluded that supply of NADPH by extramitochondrial isocitrate dehydrogenase accompanies the NADPH supply by the pentosephosphate cycle, so that in spite of the larger capacity of the latter a portion of the reducing equivalents is derived from the mitochondria via the tricarboxylates even in the fed state. This results from the necessity of relieving the inhibition of glucose‐6‐phosphate dehydrogenase by NADPH for activation of the pentosephosphate cycle.

3. Subfractionation of hepatocytes from fed rats with the digitonin method revealed decreases of the citrate/2‐oxoglutarate ratios both in the particulate (mitochondrial) and the supernatant (cytosolic) fractions, corresponding to a positive shift in the NADPH redox potential of about 10 mV (or to a decrease of the free [NADPH]/[NADP+] ratio to one‐half) in both compartments as calculated from the combined equilibria of NADP+‐linked isocitrate dehydrogenase and aconitate hydratase.

4. Only in cells from fasted, not from fed rats, the rate of aminopyrine N‐demethylation was diminished to 2/3 in the presence of aminotransferase inhibitors, suggesting a requirement for transamination steps in the transfer of NADPH‐reducing equivalents from mitochondria to cytosol under these conditions.
Limited Proteolysis of Elongation Factor Tu from Escherichia coli
Authors: JACOBSON Gary R. , ROSENBUSCH Jürg P.
Published: March 2005

Limited proteolysis of native elongation factor Tu (Mr 44000) by trypsin occurs in at least three distinct steps. The first intermediate arises through cleavage at a site about 65 residues from the amino‐terminal end of the protein. It is functionally active [Jacobson, G. R. & Rosenbusch, J. P. (1976) Biochemistry, 15, 5105–5110] and is partially protected from further degradation by the antibiotic kirromycin. The second step converts this intermediate to one of similar size (Mr 37000) which now is partially inactivated. It is likely to be identical with the intermediate described by Arai et al. [(1976) J. Biochem. Tokyo, 79, 69–83]. In the third step, the partially inactive intermediate is cleaved without any apparent change in the functional properties tested. The resulting two trypsin‐resistant fragments have molecular weights of 24000 and 14000, and remain associated under nondenaturing conditions. When either of these polypeptides, after isolation in 8 M urea, is allowed to renature, no significant reactivation of GDP binding is observed unless the isolated fragments are mixed before renaturation. These results show that the two fragments are structurally and functionally interdependent.
Ca2+ Requirement in ATP‐Induced Activation of Uncoupled Oxidation of Succinate in Isolated Rat‐Liver Mitochondria
Authors: EZAWA I. , OGATA E.
Published: March 2005

1. Small amounts of Ca2+ cause an ATP‐dependent activation of state 3 oxidation of succinate in rat liver mitochondria in vitro.

2. The Ca2+‐induced, ATP‐requiring activation of uncoupled oxidation of succinate (the Ca2+ effect) is blocked by ruthenium red, tetracaine, or high concentrations of K+. It is not blocked by La3+ or Pr3+.

3. The Ca2+ effect is demonstrable in mitochondria treated with hyposmotic shock (mitochondria devoid of the outer membrane), freezing‐and‐thawing or aging. Treatment with detergents (sodium deoxycholate and Triton X) causes a loss of the Ca2+ sensitivity.

4. For Ca2+ to produce the effect a much longer latent period (several minutes at room temperature) is needed than for ATP or succinate.

5. The Ca2+ effect is ATP dose‐related and maximum response is obtained with 0.2 mM ATP.

6. The Ca2+ effect is observed in the absence of mitochondrial accumulation of Ca2+ and is Ca2+ dose‐related with external Ca2+ between 0.5 and 50 μM. The half‐maximum response is obtained at about 1 μM.

7. The Ca2+ effect is not accompanied by an increase in the penetration of succinate across the mitochondrial membrane as judged from the swelling experiment of Chappell and Crofts, but is accompanied by the activation of succinate dehydrogenase.

8. The Ca2+ effect is demonstrable not only in the presence of external oxaloacetate or malonate, but also in conditions where endogenous oxaloacetate accumulation is prevented by the addition of rotenone and glutamate.

9. From the above results, a model is proposed in which cytosol Ca2+ alters mitochondrial metabolism by activating succinate dehydrogenase through a change of membrane conformation produced by the Ca2+ interaction with low affinity Ca2+‐binding sites on the outer surface of the inner mitochondrial membrane.
Interaction of the Phosphatidylcholine Exchange Protein with Phospholipids
Authors: WIRTZ Karel W. A. , MOONEN Peter
Published: March 2005

The phosphatidylcholine exchange protein from bovine liver has a fluorescence emission maximum at 327 nm. Fluorescence was enhanced in the presence of vesicles containing phosphatidylcholine and various amounts of either phosphatidic acid or phosphatidylglycerol. From the increase in fluorescence it was derived that the apparent dissociation constant of the exchange protein‐vesicle complex decreased with an increased vesicle content of acidic phospholipids.

Fluorescence indicated that the exchange protein interacted with lysophosphatidylcholine micelles at pH 3.5, 5.9 and 8.5. The increase in fluorescence was most prominent at the acidic pH. Circular dichroism indicated that the α‐helix content of the native protein was low between pH 3.6 and 8.0. Interaction with lysophosphatidylcholine micelles had a negligible effect on the secondary structure of the protein, except at pH 3.6 where distinct minima at 208 nm and 220 nm in the circular dichroic spectrum became apparent.
Pyrimidine Nucleosides in Solution
Authors: FOLLMANN Hartmut , PFEIL Reinhard , WITZEL Herbert
Published: March 2005

Proton magnetic resonance (PMR) spectra of 45 nucleoside and nucleotide derivatives of uracil, thymine, cytosine, 5‐methylcytosine, and other substituted pyrimidines in water or dimethyl sulfoxide solutions have been analysed in order to correlate structure variations with molecular conformations and to gain insight into the nature of intramolecular forces which are responsible for the ‘rigidity’ of the molecules. Irrespective of the structure of pyrimidine base and the absolute chemical shift values (δ) the C(6)‐H and, to a lesser degree, the C(5)‐H and C(5)‐CH3 resonances are shifted downfield by very similar amounts (Δδ) in the regular nucleosides or in the presence of charged or polar C‐4′ substituents. In detail, the following conformation‐determining effects are seen.

1. Ribose vs 2‐deoxyribose substitution of a pyrimidine makes little difference for the chemical environment of base hydrogens but an intact pentofuranose ring (4′‐O) and the exocyclic 5′‐OH group are essential for a nucleoside to assume a specific conformation.

2. Profound shielding or deshielding of H‐6 by exocyclic nitrate, halogen, hydroxyl, phosphate, and carboxylate substituents indicates preference for the anti and gauche, gauche conformation ranges in all compounds except in the case of C‐6 substitution.

3. Halogen atoms and the hydroxyl group (Δδ∼ 0.30–0.40 ppm) interact with pyrimidine bases by induced polarisation of the C(5)=C(6) double bond, phosphate anions (Δδ∼ 0.50 ppm) interact by the electrostatic attraction mechanism previously described, and in nucleoside 5′‐carboxylates (Δδ > 0.90 ppm) a hydrogen bond between −COO− and H‐6 is formed.

4. The deshielding effects caused by a 5′‐OH group and monoanionic 5′‐phosphate are similar (Δδ∼ 0.35–0.45 ppm), supporting the view that intramolecular rigidity of nucleosides and the nucleotide unit is not fundamentally different.

This systematic survey, together with our previous studies of adenosine derivatives, provides a rationale for why the majority of both pyrimidine and purine nucleosides and nucleotides in solution exhibit closely comparable conformations, which in turn explain the group specificity of certain key enzymes in nucleic acid metabolism.
Temperature‐Induced Metabolic Alterations in a Thermophilic Bacillus
Authors: REIZER Jonathan , EPSTEIN Israel , GROSSOWICZ Nathan
Published: March 2005

The effect of growth temperature on activity and rate of synthesis of the α‐aminoisobutyric acid transport system and of several enzymes of Bacillus stearothermophilus var. non diastaticus was investigated. The uptake of α‐aminoisobutyrate, as well as the activities of alanine dehydrogenase and aliphatic amidase were found to be considerably reduced in cultures grown at the near maximal temperature for growth (65°C) as compared to those grown at the optimal temperature for growth (58°C), although the growth rates at both temperatures were similar. The reduced rate at 65°C was due to a decreased differential rate of synthesis of these activities, while the increase in the activities at 58°C was due to de novo synthesis of the enzymes and the α‐aminoisobutyrate transport system rather than to activation of pre‐existing inactive enzymes or permeases. The highest rate of synthesis of alanine dehydrogenase and of the α‐aminoisobutyrate‐uptake system was obtained in cells grown at the near minimal temperature for growth (42–45°C).

The reduced rate of enzyme synthesis at the higher temperatures seemed to be due both to an increase in catabolite repression and to faulty oxidative metabolism during growth at these temperatures. The effects mentioned were only partly abolished in a mutant resistant to catabolite repression, when grown at the high temperatures. The thermoadaptation process is discussed in terms of energy production and ATP availability for protein biosynthesis.

The alterations of the metabolic activities induced by the environmental temperature were reversible and represent a physiological thermoadaptative response of the cells.
Ionization Dependence of Camphor Binding and Spin Conversion of the Complex between Cytochrome P‐450 and Camphor
Authors: LANGE Reinhard , HUI BON HOA Gaston , DEBEY Pascale , GUNSALUS Irwin. C.
Published: March 2005

The kinetic rate constants of formation and dissociation of the cytochrome‐P‐450 · camphor complex (Fe3+· RH) have been obtained by low‐temperature (+ 5°C to −20 °C) stopped‐flow experiments. Similarly the high‐spin/low‐spin equilibrium of this complex has been studied as a function of temperature and protonic activity.

Both the camphor‐binding mechanism and the high‐spin/low‐spin thermodynamic parameters of Fe3+· RH depend on the protonic activity of the medium in the physiological pH range. The binding rate constants are shown to depend on the ionization of a resdue of the protein, probably a histidine. Linear enthalpy–entropy compensation is observed for the camphor binding as well as for the spin‐state transition. A camphor‐binding‐induced change of the electrostatic potential is discussed.
Sites of Histone/Histone Interaction in the H3 · H4 Complex
Authors: BÖHM Lothar , HAYASHI Hiroaki , CARY Peter D. , MOSS Tom , CRANE‐ROBINSON Colyn , BRADBURY E. Morton
Published: March 2005

Sites of interaction between histones H3 and H4 have been probed by investigating complex formation, firstly between histone H4 and three peptides cleaved by chemical means from histone H3 (residues 1–90 and 1–120 using cyanogen bromide and residues 42–135 using N‐bromosuccinimide), secondly between histone H3 and two peptides cleaved from histone H4 (residues 1–84 using cyanogen bromide and residues 38–102 using chymotrypsin) and thirdly between the H4 peptide (residues 38–102) and the three H3 peptides (residues 1–90, 1–120 and 42–135).

The criterion for complex formation is the appearance of characteristic perturbed resonances in the aromatic region of the 270–MHz proton resonance spectrum of the peptide mixture. It is concluded that loss of 37 N‐terminal residues from histone H4 and 41 N‐terminal residues from histone H3 does not prevent complex formation, whilst the loss of 18 C‐terminal residues from H4 and 45 C‐terminal residues from H3 does prevent it; the last 15 C‐terminal residues of H3 are, however, not required for forming a complex. The regions important for complex formation are therefore defined as residues 42–120 in histone H3 and residues 38–102 in histone H4.
Phosphorylation of the Calcium‐Transport Adenosine Triphosphatase of Cardiac Sarcoplasmic Reticulum by Orthophosphate
Authors: WINKLER Franz , SUKO Josef
Published: March 2005

Membranes of sacroplasmic reticulum prepared from dog hearts are phosphorylated by [32P]‐orthophosphate in the presence of a calcium load and magnesium. The [32P]phosphate incorporation into sarcoplasmic reticulum follows Michaelis‐Menten kinetics with an apparent Michaelis Constant of 0.5 mM for orthophosphate (pH 7.0). The phosphorylation is pH‐dependent, with an optimum pH of 6.0–6.2 (maximum phosphoprotein steady state level 0.6–0.8 nmol/mg protein).

The phosphorylation of sarcoplasmic reticulum by [32P]orthophosphate is strongly inhibited by calcium in the medium. Half‐maximum inhibition occurs at an ionized calcium concentration of about 0.8 μM. The phosphoprotein steady‐state level is reduced by 85–90% by phospholipase‐A treatment or solubilization of calcium‐preloaded sarcoplasmic reticulum with Triton X‐100.

The phosphoprotein of sarcoplasmic reticulum formed from [32P]orthophosphate is dephosphorylated by ADP with resultant in ATP synthesis. Phosphoprotein formation and ATP synthesis by sarcoplasmic reticulum are unaltered by azide, dinitrophenol, dicyclohexyl carbodiimide and oligomycin.

The phosphoprotein is acid‐stable. The trichloroacetic‐acid‐denaturated 32P‐labelled membrane complex is dephosphorylated by hydroxylamine, indicating that the phosphorylated protein is an acylphosphate.

Polyacrylamide gel electrophoresis (performed with phenol/acetic acid/water) of sarcoplasmic reticulum phosphorylated by [32P]orthophosphate demonstrates that the phosphate incorporation occurs into a protein with a molecular weight of about 100 000, as in the case of phosphoprotein formation from ATP.

The data suggest that the phosphoprotein of the calcium‐dependent ATPase formed from inorganic phosphate represents a high‐energy intermediate of the reverse reaction of the calcium pump of cardiac sarcoplasmic reticulum.