Substrate specificity of copper-containing plant amine oxidases

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Folin试剂制备中的一个改进

本文介绍了Folin试剂制备中的一个改进,使制备过程得到简化.     

On the specificity of porcine elastase.

The specificity of porcine elastase (EC 3.4.4.7) has been studied. Ethyl esters derived from benzoyl amino acids with straight side chains are better substrates than those with branched side chains; the best substrate is norvaline ester. In the series of benzoylalanine alkyl esters the alcohol moiety markedly affects the susceptibility. The benzyl ester was found to be the best nonactivated substrate derived from monomeric amino acid. With elastase acylation is rate limiting, in contrast to chymotrypsin and trypsin where deacylation is generally the rate determining step with specific ester substrates.     

Base and conformational specificity of an amine modification of DNA

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A-form, Z-form, or the single-strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH2O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of adduct formation under standard reaction conditions (27 degrees C, 0.63M CH2O, 0.007M n-butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI-dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG-dC) show enhanced 2'-endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA-dT) in the "X"-form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23, 1-4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B-form of polynucleotides as demonstrated by the failure of poly(dG-m5dC) in the stable Z-form to exhibit substantial reaction. The B-form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta-lactamase-catalyzed aminolysis of depsipeptides: amine specificity and steady-state kinetics

beta-Lactamases catalyze not only the hydrolysis but also the aminolysis of certain depsipeptides [Pratt, R. F., & Govardhan, C. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302-1306]. This paper explores further the specificity of the aminolysis reaction with respect to the structure of the amine and also the steady-state kinetics of the reaction. The amines preferred by the class C beta-lactamase of Enterobacter cloacae P99 appear to be aromatic D-alpha-amino acids. The general order of substrate effectiveness at pH 7.5 appears to be aromatic D-alpha-amino acids greater than large aliphatic D-alpha-amino acids greater than small aliphatic D-alpha-amino acids approximately small aliphatic L-alpha-amino acids greater than large L-alpha-amino acids. Charges on the aliphatic side chains seem unimportant. Ineffective as acyl acceptors were beta-amino acids, alpha-amino phosphonic acids, and, in general, amines, including amino acid carboxyl derivatives and peptides. There is thus strong evidence for specific interaction between the amine and the enzyme. A detailed kinetics study was made of the P99 beta-lactamase-catalyzed aminolysis of m-[[(phenylacetyl)glycyl]oxy]benzoic acid by D-phenylalanine. The steady-state kinetics were complex because of the presence of parallel enzyme-catalyzed hydrolysis and aminolysis reactions. An empirical rate equation was obtained for the total reaction. This has important elements in common with that previously found for the aminolysis of specific peptides by the DD-peptidases of various Streptomyces strains [e.g., Frere, J.-M., Ghuysen, J.-M., Perkins, H.R., & Nieto, M. (1973) Biochem. J. 135, 483-492].(ABSTRACT TRUNCATED AT 250 WORDS)     

A procedure for eliminating interferences in the lowry method of protein determination

A modification of the Lowry assay for the quantitative protein measurement in the presence of interfering materials has been developed. The method is based on a precipitation with a single-phase hexane:isopropanol solvent system and later resuspension of protein pellets with sodium dodecyl sulfate and deoxycholate. The new procedure eliminates the interference caused by Triton X-100, phospholipids, or dithiothreitol providing yields higher than 95% and seems to be especially suitable for protein determination on membrane preparations in samples with small volumes and/or very low protein concentrations.     

Hydrophilic fluorescein derivatives: useful reagents for liposome immunolytic assays

Hydrophilic derivatives of fluorescein containing hydroxyalkyl substituents were synthesized and encapsulated within liposomes. The fluorophores showed significantly more retention with time than did fluorescein, carboxyfluorescein, or calcein. Unlike calcein, the fluorophores are minimally susceptible to fluorescence quenching by Co2+. The utility of these compounds as immunodiagnostic reagents was demonstrated by encapsulating 5(6)-carboxyfluorescein trismethylolamide in haptenized liposomes which were used in an immunoassay for digoxin.     

On the heterogeneity and organ specificity of chicken tropomyosins.

1. Tropomyosins from chicken cardiac, skeletal, and gizzard muscles were each resolved into two subunits by polyacrylamide gel electrophoresis in a system containing sodium dodecylsulfate (SDS), urea and sodium borate, and were designated C1 C2, S1 S2, and G1 G2, respectively, in descending order of mobility on electrophoresis. S1, S2, G1, and G2 were prepared as pure samples by electrophoresis. 2. The apparent molecular weights of C (C1 + C2), S1, S2, G1, and G2 were calculated to be 36,000, 36,000, 37,500, 36,000, and 40,000, respectively, based on SDS gel electrophoretic mobility according to the method of Weber and Osborn. C and S1 showed nearly the same mobility in all electrophoretic systems tried. S1 and G1, which comigrated in an SDS-sodium borate system, showed different mobilities upon addition of 5 M urea to the system. 3. Immunological evidence presented indicates that each subunit has a specific antigenic site(s) in addition to an identical one(s) in common with the others. 4. As each tropomyosin subunit formed two precipitin lines with the homologous antiserum, as many as ten kinds of subunits may exist in chicken muscles.     

On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase.

Data are presented that support a nonenzymic mechanism for the staining obtained with the cobalt-bicarbonate method. The biochemically inactive apocarbonic anhydrase and Cu+2 apocarbonic anhydrase stain positively and this stain is inhibited by acetazolamide. The staining of the acetazolamide resistant carbonic anhydrase of male rat liver is inhibited by 10-6 M acetazolamide, at which concentration no biochemical inhibition is observed. There is no correlation between the biochemical and histochemical inhibitory potencies of a number of sulfonamides. The nonsulfonamide inhibitor, KCNO, does not inhibit staining. When incubations are performed in media exposed to atmospheres of increasing CO2 content, staining is not abolished until the atmospheric pCO2 approaches that generated by the medium itself. This finding renders the carbonic anhydrase catalyzed dehydration of HCO3- an improbable reaction for the staining. Studies with modified media show differences in staining patterns and in sensitivity to acetazolamide inhibition which question the specificity of the method for carbonic anhydrase.     

Attachment of protein affinity-labeling reagents of variable length and amino acid specificity to E. coli tRNAfMet.

Transamination with bifunctional amines in the presence of bisulfite has been used to attach side chains of variable length to the N4-position of single stranded cytidine residues in E. coli tRNAfMet. Such side chains, terminating in reactive primary amino groups, have been coupled to a variety of N-hydroxysuccinimide esters. The resulting modified tRNAs carry protein affinity labeling groups capable of covalent reaction with a variety of amino acids.