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At least 19 recordsLinked to original sources
PubMed1977

Identification of a pyruvoyl residue in S-adenosylmethionine decarboxylase from Saccharomyces cerevisiae.

S-Adenosylmethionine decarboxylase from Saccharomyces cerevisiae has been purified to homogeneity. Acid hydrolysis of NaB3H4-reduced enzyme released 2.2 mol of tritiated lactate per mol of dimeric enzyme, indicating that a pyruvate moiety is present. Inhibition of enzymatic activity by NaBH4 reduction and by carbonyl-binding reagents indicates that this pyruvoyl residue is required for the activity of the enzyme. This is the first example reported of a eukaryotic enzyme containing a covalently linked pyruvoyl residue.

Adenosylmethionine Decarboxylase↗
PubMed1978

Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.

The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.

Avian Leukosis Virus↗
PubMed1979

Cross-reactivity of rat, mouse, and human IgD.

Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.

Animals↗
PubMed1977

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.

Bacterial Proteins↗
PubMed1979

Some molecular properties of NAD(P)H dehydrogenase from rat liver.

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same K(m) value for NADH but different values for V(max.) were obtained for the enzyme purified from Sprague-Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.

Amino Acids↗
PubMed1975

Mercuri-nitrophenol as a reporter group for the conformational change of hemoglobin.

One mole of horse hemoglobin tetramer reacts with 2 moles of 2-chloromercuri-4-nitrophenol (MNP) at beta 93 cysteine. The difference spectra between NMP-bound hemoglobin and hemoglobin, measured with the aid of ascorbic acid and ascorate oxidase [EC 1.10.3.3] as deoxygenation reagents, indicate that the pK of the phenolic hydroxyl group of MNP increases by 0.6 to 0.8 pH unit on deoxygenation of the hemoglobin. The Hill constant of the modified hemoglobin changes with pH. It decreases from about 2.4 at pH 6.8 to about 1.0 at pH 9.0 This effect of the reagent is interpreted as inherent to the reporter groups.

Animals↗
PubMed1976

A kinetic analysis of a catechol-specific binding site in the microsomal fraction from the rabbit aorta.

(-)-3/-Norepinephrine (3H-NE) binding to the microsomal fraction of the rabbit aorta has been studied. Binding appears to increase linearly with time up to at least 30 min, shows no evidence of stereoselectivity and may be inhibited only by compounds possessing the catechol or 3-methoxy-4hydroxyphenyl moieties, with the latter being 100-fold less effective. 3H-NE binding is saturable with a Km of 8.5 X 10(-8) M and V max of 28 pmoles/mg protein. A Hill plot indicates that binding is noncooperative whereas a Scatchard plot suggests that two sites may be present. Binding does not appear to require physiological concentrations of Ca2+ or Mg2+ and is inhibited significantly by EDTA and sodium metabisulfite. In addition, binding is markedly enhanced by low and high pH values. This binding is also inhibited by sodium metabisulfite which suggests that an oxidized form of the catecholamine is the active binding species. Experiments with several group specific reagents indicate that binding may require a free sulfhydryl group but not a carboxyl function. The binding process requires an energy of activation of 14.8 kcal/mole whose magnitude may be partly explained, with the aid of optical rotatory dispersion spectra, by a non-stereoslective conformational change in protein structure induced by the amine. The characteristics of the 3H-NE binding sites observed in the microsomal fractional of the rabbit aorta appear to be different from those expected if binding were to the adrenoreceptors. A possible mechanism for catecholamine binding to free sulfhydryl groups on protein is presented.

Adrenergic beta-Antagonists↗
PubMed1976

Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes.

Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents. The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists; strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000. Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8 X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA. Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding. These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties.

Adrenergic beta-Antagonists↗
PubMed1979

Modification of human serum albumin with N-(2,5-dinitro-4-fluorophenyl)-4-amino-2,2,6,6-tetramethyl-piperidinooxy radical.

Human serum albumin has been treated with the spin-labeling reagent indicated in the title. Ultraviolet spectral studies of the protein so modified suggest that reaction takes place at lysine and tyrosine sidechains; kinetic experiments indicate that there are two especially reactive amino groups of the protein which are preferentially modified. Evidence is presented that these groups include the one acetylated by aspirin (Lys-199) or those arylated by 2.6-dinitro-4-trifluoromethylbenzenesulfonate. Esr experiments show that bound spin labels have about the same correlation time expected for overall tumbling of the protein; ESR observations indicate that molecular freedom near the spin labels is not increased when the protein is transferred to 8 M urea.

Binding Sites↗
PubMed1979

Stability of a reference panel of lyophilized hepatitis B antigens and antibodies.

A lyophilized hepatitis B working/reference panel has been prepared for use in standardization tests. This panel includes HBsAg, anti-HBs, HBeAg/anti-HBe and subtype reagents. Quantitative analysis of the HBsAg reagents indicates that at a storage temperature of -20 degrees C, only 1 log at maximum of RIA counts per minute would be lost in 95 years. After storage at -20 degrees C for 1 year, there has been no loss of reactivity in any of the tests used to detect HBsAg, anti-HBs, HBeAg/anti-HBe or subtypes.

Antibodies, Viral↗
PubMed1979

Consumption of C3 via the classical and alternative complement pathways by sera and synovial fluids from patients with rheumatoid arthritis.

Five sera and four synovial fluids from patients with rheumatoid arthritis were found to contain substances which consumed hemolytic C3 in normal human serum (NHS) and in normal guinea pig serum (NGPS). These fluids were then tested for ability to activate the alternative pathway by incubating them with NHS containing MgEGTA and C4 deficient GPS. All sera and two synovial fluids depleted C3 in these reagents, indicating direct activation by the alternative pathway. Density gradient ultracentrifugation demonstrated C3 fixing activity in some specimens in the greater than 19s regions. These substances may also activate complement similarly in vivo and participate in the development of inflammatory processes associated with this disease.

Adult↗
PubMed1977

Assay of serum free fatty acids by extraction-photometric procedure.

Reaction conditions for extraction-photometric determination of free fatty acids in serum were studied. The results were found to be influenced by (a) sodium chloride concentration in the copper reagent, (b) the kind of standard solution used, (c) centrifugation of the reaction mixture and (d) serum phospholipids. A micromethod based on the formation of free fatty acids-copper soaps with a stable copper reagent and sensitive indicator for copper, 2-(2-thiazolylazo)-4-methoxyphenol, is described.

Copper↗
PubMed1975

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.

Adenosine Monophosphate↗
PubMed1979

Nitration of polypeptides using ethanol in reaction buffers minimizes crosslinking.

High concentrations of ethanol are effective in reducing the intermolecular cross-linking which predominates when the reactions of tetranitromethane with the carboxypeptidase inhibitor from potatoes, a chymotrypsin inhibitor also from potatoes, and insulin are carried out under standard conditions. Including reagents such as ethanol may be of general utility in the preparation of monomeric nitro-derivatives of hydrophobic, low-molecular-weight proteins.

Buffers↗
PubMed1975

Superactivation of thermolysin by acylation with amino acid N-hydroxysuccinimide esters.

Synthesis of a series of active N-hydroxysuccinimide esters of aliphatic and aromatic amino acids has yielded a new class of reagents for the covalent modification of proteolytic enzymes such as thermolysin. The activities of aliphatic acyl amino acid thermolysins are from 1.7 to 3.6 times greater than that of the native enzyme when hydrolyzing durylacryloyl-Gly-Leu-NH2, the substrate employed most widely. By comparison, the aromatic acylamino acid derivatives are "superactive," their activities being as much as 70-fold greater. Apparently, the aromatic character of the amino acid introduced is a critical variable in the determination of the functional response. The increased activity is completely restored to that of the native enzyme by deacylation with nucleophiles, such as hydroxylamine, and the rate of restoration of native activity is a function of the particular acyl group incorporated. Preliminary evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified. The functional consequences of successive modification with different reagents, moreover, indicate that each of them reacts with the same protein residue. The competitive inhibitors beta-phenyl-propionyl-Phe and Zn-2+ do not prevent modification with these active esters. Hence, the site(s) of their inhibitory action differ(s) from that at which modification occurs. The structure of the substrate is also a significant variable which determines the rate at which each acyl amino acid thermolysin hydrolyzes peptides. Depending on the particular substrate, the activity of aromatic derivatives can be as much as 400-fold greater than that of the native enzyme, and the resultant activity patterns can be ordered in a series characteristic for each enzyme derivative.

Acylation↗
PubMed1976

Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase.

After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate.

Animals↗
PubMed1979

Determination of total CO2 in plasma by automated flow-injection analysis.

We describe a procedure for measuring total CO2 in plasma, based on the principles of the flow-injection analysis technique, which makes use of unsegmented fast-flowing reagent streams, as developed by Růziĉka and co-workers. The further methodological design resembles the silicone-rubber membrane technique of Kenny and Cheng. CO2 in the sample is released by reaction with H2SO4. Appropriate amounts of CO2 permeate through the membrane that separates the acid reagent streams and a buffered cresol-red indicator system. The experimental set-up and functioning of this system are described.

Autoanalysis↗