Cleavage of active peptides by a lung enzyme

Zusammenfassung Ein Enzym (Angiotensin I «converting enzyme» oder Kininase II; DH) wurde aus Schweinelungen isoliert und gereinigt. DH splatet Dipeptide vom Carboxyterminus der Peptidsubstrate mit Einschluss von Bradykinin, Angiotensin I, B-Kette von Insulin und¹⁴C-DNS-Gly-Gly-Gly. Zu den besten Inhibitoren des Enzyms gehören zwei kürzlich synthetisierte Peptide sowie Glutathion und Insulin. Die Experimente deuten auf ein einziges Enzym hin, das alle erwähnten Substrate hydrolysiert.

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Supported in part by grants No. HE 08764 and No. 5T01 HE 05859 from N.I.H., U.S.P.H.S. and by the O.N.R. No. N00014-68-A-0496 and No. N00014-69-A-0385.

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During the tenure of a fellowship of the Oklahoma Heart Association.

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Acknowledgements. MissDeborah Downs assisted us in some of the experiments. HHL was donated by Dr.D. Cushman of the Squibb Institute.     

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex

Summary Fast and very slow hydrolyses of des-Arg⁹-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, K_m values, for des-Arg⁹-bradykinin and bradykinin were found to be 0.24 mM and 4.4 M, and the maximum velocities, V_max values (mol·min^–1·mg protein^–1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.Acknowledgments. We thank Dr Kinzo Nagasawa of Kitasato University for encouragement in this work and Dr Ervin G. Erdös, University of Texas, Health Science Center, Dallas, for valuable discussion and suggestions.Abbreviations used: Z, carbobenzoxy; Bz, benzoyl; ACE, angiotensin I-converting enzyme; BK, bradykinin; HPLC, high performance liquid chromatography.     

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex

Fast and very slow hydrolyses of des-Arg9-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, Km values, for des-Arg9-bradykinin and bradykinin were found to be 0.24 mM and 4.4 microM, and the maximum velocities, Vmax values (mumol . min-1 . mg protein-1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.     

Inhibition of angiotensin-converting enzyme by analogs of peptides fromBothrops jararaca venom

Zusammenfassung Nachweis mit Analogen der im Gifte vonBothrops jararaca gefundenen Peptide, dass die Inhihibition des Angiotensins «converting enzyme» von zwei eindeutigen Teilsequenzen dieser Peptide abhängig ist. Die hohe spezifische kompetitive Inhibition, hervorgerufen durch die Peptide vonBothrops jararaca, wird der Bindung ihrer Tripeptidreste vom Carboxyterminus mit dem aktiven Zentrum des Enzyms zugeschrieben, die in gleicher Weise wie die Peptidsubstrate mit dem Enzym gebunden werden. Die Wirksamkeit der Giftpeptide hängt von der Bindung eines zweiten Teiles der Peptide mit dem Enzym ab.     

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming α-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3₁₀/α helix, whereas the shortest peptides tend to adopt a β-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes. Received 26 January 2001; received after revision 7 March 2001; accepted 15 March 2001     

Further applications of pentachlorophenyl active esters in the synthesis of peptides

Zusammenfassung Pentachlorophenylaktivierte Ester von N-Carbobenzoxy-aminosäuren verbinden sich in guten Ausbeuten mit Dicyclohexylaminsalzen von Aminosäuren. Um die Hydrolyse zu vermeiden, wurde eine Kombination der Methoden der gemischten Anhydriden und pentachlorophenylaktivierten Estern zur Verlängerung der Peptidenketten benützt.     

Modification of effects of biologically active peptides,caused by enzyme treatment,on the excitability of identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

Summary Physalaemin, which excites an identifiable molluscan giant neurone (the TAN, tonically autoactive neurone), lost the effect after the trypsin treatment. Unexpectedly, this peptide shows an inhibitory effect on the same neurone after chymotrypsin treatment. Deamino-dicarba-(d-d-)oxytocin and d-d-Arg-vasotocin, which excite another identifiable neurone (the PON, periodically oscillating neurone) continue to show the effect after chymotrypsin treatment (6 h). But d-d-Arg-vasotocin losts the effect on the PON after trypsin treatment.The authors thank Dr Sadaaki Iwanaga of Osaka University and Dr Atsuo Inoue of Daiichi Pharmaceutical Co. for their helpful advice, and Miss Hiroko Tamura for her technical assistance.     

Enhancement of humoral immune response against human lung elastin peptides

Summary When guinea pigs instead of rabbits were used as the host animals, 8–16 times higher antibody titers against human lung elastin peptides were produced with only 1/20 the amount of antigen per unit body weight. This corresponds to a 200-fold enhancement of the immune response.Presented at the 6th Colloquium of the Federation of European Connective Tissue Clubs, Paris, August 28–30, 1978. The data form part of the Ph.D. thesis submitted by T.V. Darnule to the Department of Biology, New York University. Supported in part by NIH Program Project Grant HL15832 and by a Parker B. Francis Fellowship to T.V. Darnule.     

Inhibition of mushroom tyrosinase by 3-amino-L-tyrosine: molecular probing of the active site of the enzyme

We report the ability of 3-amino-L-tyrosine to act as a fully reversible competitive inhibitor of mushroom tyrosinase. The inhibition is linked to the ortho-aminophenol structure, and a copper bridging mechanism in the active site is proposed.     

Inhibition of mushroom tyrosinase by 3-amino-L-tyrosines: Molecular probing of the active site of the enzyme

Summary We report the ability of 3-amino-L-tyrosine to act as a fully reversible competitive inhibitor of mushroom tyrosinase. The inhibition is linked to the ortho-aminophenol structure, and a coppe bridging mechanism in the active site is proposed.     

Mutual replaceability of amide and ester groups in biologically active peptides and depsipeptides

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Zinc deficiency and lung converting enzyme activity in rats

Summary Angiotensin converting enzyme activity was found to be significantly decreased in the isolated perfused lung from zinc-deficient rats when compared with that of controls. Addition of zinc ion to the superfusion medium did not cause a recovery in this decreased activity of the enzyme. It is postulated that zinc deficiency probably produces a structural change in the lung angiotensin converting enzyme.This work is supported by a grant from Turkish Scientific and Technical Research Council (TAG-350).     

Polymerization of phenolic intermediates of pesticides by a fungal enzyme

Summary The fungusRhizoctonia praticola produces an extracellular phenol oxidase (laccase) which polymerizes phenolic intermediates of various pesticides. The enzyme catalyzes the formation of oligomeric products from halogenated phenolic intermediates of phenoxyalkanoate herbicides and from naphtholic products derived from carbamate insecticides. These findings permit further investigations into the mechanism and role of oxidative coupling leading to the incorporation of xenobiotic compounds into soil organic matter.     

Zinc deficiency and lung converting enzyme activity in rats.

Angiotensin converting enzyme activity was found to be significantly decreased in the isolated perfused lung from zinc-deficient rats when compared with that of controls. Addition of zinc ion to the superfusion medium did not cause a recovery in this decreased activity of the enzyme. It is postulated that zinc deficiency probably produces a structural change in the lung angiotensin converting enzyme.     

Mapping of restriction enzyme cuts by a new two-dimensional procedure

A new procedure has been worked out to establish restriction maps. The method is fast, does not in general require labeled DNA and has been applied to map the linear palindromic rDNA of Physarum with the restriction enzyme Bst EII.     

Mapping of restriction enzyme cuts by a new two-dimensional procedure

Summary A new procedure has been worked out to establish restriction maps. The method is fast, does not in general require labeled DNA and has been applied to map the linear palindromic rDNA ofPhysarum with the restriction enzymeBstEII.Acknowledgment. This work was supported by grants from the NIH (GM-30338), the NSF (PCM 8116391) and the Swiss National Science Foundation (3.075.81).     

Quantification of digoxin by enzyme immunoassay: synthesis of a maleimide derivative of digoxigenin succinate for enzyme coupling

Summary We synthesized the m-maleimidobenzoyl derivative of digoxigenin-3-0-succinate (through a p-phenylenediamine bridge) as a hapten derivative directed towards coupling to sulfhydryl groups of -galactosidase. Prepared enzyme conjugate had about 97% of the enzyme labeled with the hapten derivative while retaining full enzyme activity. The enzyme immunoassay for digoxin we prepared showed a maximum sensitivity of 30 pg per assay (c.v.=3%) with minimal cross-reaction with digotoxin (3.8%). Our method for hapten conjugation to -galactosidase is highly efficient and is simple and easily replicated.     

Polymerization of phenolic intermediates of pesticides by a fungal enzyme.

The fungus Rhizoctonia praticola produces an extracellular phenol oxidase (laccase) which polymerizes phenolic intermediates of various pesticides. The enzyme catalyzes the formation of oligomeric products from halogenated phenolic intermediates of phenoxyalkanoate herbicides and from naphtholic products derived from carbamate insecticides. These findings permit further investigations into the mechanism and role of oxidative coupling leading to the incorporation of xenobiotic compounds into soil organic matter.