Hoppe-Seyler's Zeitschrift fur physiologische Chemie最新文献

An adipokinetic hormone was purified from corpora cardiaca of au adult Manduca sexta (tobacco hornworm)by reversed-phased high performance liquid chromatography. The following amino-acid composition was determined: 2 Thr, 2 Ser, Glx, Gly, Leu, Phe, Trp. The adipokinetic activity was tested in adult Manduca. In a bioassay in which activation of fat body glycogen phosphorylase was measured in ligated larvae, the hormone elicited a maximal response at the same concentration as it did in the bioassay for hyperlipaemic activity.

By means of a sensitive electrophoretic technique for the detection of proteinase inhibitors three slowly migrating proteinase inhibitors (SMPI) were discovered in some samples of pathological urine. SMPI 1 migrated in the beta 2-zone whereas SMPI 2 and SMPI 3 appeared in the anodal and cathodal gamma-zone, respectively. Only SMPI 1 and 2 were examined in detail. These were found to inhibit tryptic and elastolytic digestion, but not chymotryptic or plasminolytic digestion of casein. Immunological investigations revealed no similarity to normally occurring proteinase inhibitors in serum and urine. The SMPIs from one sample of urine were partially purified by DEAE-Sephadex ion exchange chromatography, followed by gel filtration on Sephacryl superfine 200. This procedure did not separate the two inhibitors. The molecular masses were estimated to be 25 000 Da by gel filtration, and 23000-26500 Da by SDS polyacrylamide gel electrophoresis.

Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).

The effect of gabexate mesilate (FOY) was studied in vitro in human and canine serum upon the addition of trypsin, and in vivo in dogs during intravenous trypsin infusion. The effect of FOY was compared with the effect of aprotinin. FOY did not show any protection against trypsin-induced activation of the complement and kinin systems in vitro or in vivo, while aprotinin did. All dogs exhibited signs of circulatory shock together with a consumption of the two main proteinase inhibitors, alpha-macroglobulin and alpha 1-proteinase inhibitor, when intravenous infusions of FOY and trypsin were performed simultaneously. However, all dogs survived without signs of shock if aprotinin was given instead of FOY. The ineffectiveness of FOY in serum is explained by the complete dissociation of FOY trypsin complexes together with a rapid degradation of FOY to inactive metabolites. Although FOY is an effective proteinase inhibitor in defined buffer systems in vitro, the results of the present study indicate that it is not an effective proteinase inhibitor in vivo. Aprotinin protects against trypsin-induced activation reactions, although much higher concentrations are needed in human than in canine serum.

The inhibition of acetylcholinesterase with fluorophores type II and III linked with the OH-selective fluorophosphonoyl groups is kinetically investigated by comparing the changes in activity and fluorescence. The hydrolysis of the fluorescent phosphonoylfluorides 1 and 4 to 7 in aqueous buffer solutions does not interfere with the inhibition kinetics. The inhibition constants of the investigated compounds are unexpectedly high (10(6) to 10(8) S-1M-1). They increase with increasing spacer length, but arrive at an optimal value with four methylene groups in the inhibitor 6. The fluorescence is quenched by the interaction of 6 and acetylcholinesterase. This fact can be used for the determination of acetylcholinesterase by fluorescence titration (Fig. 9). Fluorescence once more increases slowly during the aging process, leading to the degradation products 9, 11 and 12. In acetylcholinesterase inhibited by 1, a sensitized fluorescence is observed, produced by tryptophane intrinsically linked to the esterase. In the presence of quaternary ammonium salts like acetylcholine chloride or gallamine triethochloride 15, the decrease of fluorescence is lower. Acetylcholinesterase inhibited in this way is reactivated quantitatively by toxogonine. No reactivation is possible with acetylcholinesterase inhibited in the absence of the above mentioned quaternary ammonium salts. As a result of the investigation using fluorescent inhibitors the conclusion can be drawn that not only the active site of acetylcholinesterase is blocked by phosphonoylation. The conformation too seems to be influenced by interactions of the inhibitors with the hydrophobic areas of the enzyme.

The amino-acid sequence and the arrangement of the disulfide bonds of the human free secretory component were completely elucidated by the methods of protein chemistry. The free secretory component is a monomeric glycoprotein (Mr approximately 86000), consisting of 558 amino acids with 7 carbohydrate chains bound to asparagine. The protein contains 20 cysteine residues but, as a special feature, no methionine. The polypeptide chain is divided into five regions of internal homology, 104 to 114 amino acids in length. The 20 cysteine residues form 10 disulfide bonds, 9 of which confirm the internal homology by their characteristic arrangement. The free secretory component also shows homology to immunoglobulins in some sections. A computer-supported tertiary structure is proposed for the free secretory component.

Antibody 50S10.1 is a hybridoma-derived gamma 3 kappa antibody of BAB-14 mouse strain origin, with specificity for N-acetylglucosamine beta 1----3 linked to L-rhamnose, the immunodeterminant of the streptococcal Group A polysaccharide. The VL50S10.1 amino acid sequence is the fourth complete one reported with this specificity and the first fully determined V kappa 21A structure. Furthermore it is the first V kappa 21A isotype sequence derived from an antibody with known antigen specificity. The V kappa region of this and the previously described monoclonal anti-streptococcal Group A polysaccharide antibodies 7S34.1, 2S1.3 and 17S29.1 are compared, showing that in monoclonal antibody 50S10.1 a V kappa germline gene is expressed which is unrelated to those previously shown to be expressed in antibodies of this specificity. V kappa 50S10.1 increases the variability of known murine V kappa regions and confirms stretches of V kappa 21A sequences previously established.

beta-Lactoglobulin-like proteins were detected in horse colostrum and normal milk using immunological techniques. In contrast to the beta-lactoglobulins sequenced so far these proteins are monomeric and genetically not homogenous. In this paper we report the first primary structure of a monomeric beta-lactoglobulin from horse colostrum. By means of an automatic liquid-phase sequenator the sequence of peptides obtained by tryptic digestion and by cyanogen bromide cleavage was determined. A limited tryptic digestion and hydrolysis with chymotrypsin provided the necessary overlapping peptides. The horse beta-lactoglobulin I consists of 162 amino acids, among these four cysteine, six methionine residues and one tryptophan residue. Homologous comparison with bovine beta-lactoglobulin A shows an unexpectedly great difference of 72 amino acids (or 44%). Thirteen of these exchanges are explained as two-point mutations. We found that the free thiol group, localized at position 121 or in equal amounts at positions 119 and 121 in bovine beta-lactoglobulin, is absent in beta-lactoglobulin I from horse colostrum. In position 121 a tyrosine substitution for cysteine was found. The amino-acid exchanges of the horse beta-lactoglobulin I as compared to the other beta-lactoglobulins are discussed.