[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase

S. M. Strittmatter, D. R. Lynch, Solomon H Snyder

Research output: Contribution to journalArticle

Abstract

[3H]guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a K(D) of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu]enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H]GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their K(i) values against [3H]GEMSA binding are similar to their K(i) values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the V(max) for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the B(max)(maximal number of binding sites) for [3H]GEMSA is about 20,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H]GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the K(i) of GEMSA against enzymatic hydrolysis and weakly inhibits [3H]GEMSA binding by increasing the K(D).

Original languageEnglish (US)
Pages (from-to)11812-11817
Number of pages6
JournalJournal of Biological Chemistry
Volume259
Issue number19
StatePublished - 1984

Fingerprint

Carboxypeptidase H
Labeling
Tissue
Brain
Enzymes
guanidinoethylmercaptosuccinic acid
Leucine Enkephalin
Affinity chromatography
Methionine Enkephalin
Acids
Enzymatic hydrolysis
Enzyme activity
Pituitary Gland
Affinity Chromatography
Edetic Acid
Purification
Arginine
Rats
Assays
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase. / Strittmatter, S. M.; Lynch, D. R.; Snyder, Solomon H.

In: Journal of Biological Chemistry, Vol. 259, No. 19, 1984, p. 11812-11817.

Research output: Contribution to journalArticle

@article{357bd9a3896b45078e8529308104069c,
title = "[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase",
abstract = "[3H]guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a K(D) of 6 nM. Specific binding accounts for greater than 95{\%} of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu]enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H]GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their K(i) values against [3H]GEMSA binding are similar to their K(i) values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the V(max) for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the B(max)(maximal number of binding sites) for [3H]GEMSA is about 20,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H]GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the K(i) of GEMSA against enzymatic hydrolysis and weakly inhibits [3H]GEMSA binding by increasing the K(D).",
author = "Strittmatter, {S. M.} and Lynch, {D. R.} and Snyder, {Solomon H}",
year = "1984",
language = "English (US)",
volume = "259",
pages = "11812--11817",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

TY - JOUR

T1 - [3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase

AU - Strittmatter, S. M.

AU - Lynch, D. R.

AU - Snyder, Solomon H

PY - 1984

Y1 - 1984

N2 - [3H]guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a K(D) of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu]enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H]GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their K(i) values against [3H]GEMSA binding are similar to their K(i) values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the V(max) for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the B(max)(maximal number of binding sites) for [3H]GEMSA is about 20,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H]GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the K(i) of GEMSA against enzymatic hydrolysis and weakly inhibits [3H]GEMSA binding by increasing the K(D).

AB - [3H]guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a K(D) of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu]enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H]GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their K(i) values against [3H]GEMSA binding are similar to their K(i) values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the V(max) for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the B(max)(maximal number of binding sites) for [3H]GEMSA is about 20,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H]GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the K(i) of GEMSA against enzymatic hydrolysis and weakly inhibits [3H]GEMSA binding by increasing the K(D).

UR - http://www.scopus.com/inward/record.url?scp=0021284313&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021284313&partnerID=8YFLogxK

M3 - Article

C2 - 6434531

AN - SCOPUS:0021284313

VL - 259

SP - 11812

EP - 11817

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -