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| IDENTIFICATION OF REACTIVE VICINAL CYSTEINES IN SACCHAROMYCES-CEREVISIAE (ATP) AND SYSTOLIC RAT-LIVER (GTP) PHOSPHOENOLPYRUVATE CARBOXYKINASES |
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| Author(s): ROJAS MC, ENCINAS MV, KEMP RG, LATSHAW SP, CARDEMIL E |
| Source: BIOCHIMICA ET BIOPHYSICA ACTA Volume: 1164 Issue: 2 Pages: 143-151 Published: JUL 10 1993 |
| Times Cited: 14 References: 33 |
| Abstract: Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phosphoenolpyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide. Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2. Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated. The fluorescence spectra of the labeled proteins showed the pyrene excimer emission band. The pyrenyl-derivatized enzymes were digested with trypsin after carboxymethylation, and two labeled peptides were isolated for each carboxykinase upon reverse-phase high-performance liquid chromatography. Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzyme), and cysteines 288 and 413 (rat enzyme) were labeled with the fluorescence SH-specific reagent. The relative reactivity of these residues was characterized. Labeling experiments utilizing the 5,5'-dithiobis(2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-groups occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV SANTIAGO, FAC CIENCIA, DEPT QUIM, CASILLA 307, SANTIAGO 2, CHILE
2. UNIV SANTIAGO, FAC CIENCIA, DEPT QUIM, SANTIAGO, CHILE
3. UNIV HLTH SCI CHICAGO MED SCH, DEPT BIOL CHEM, N CHICAGO, IL 60064 USA |
| Publisher: ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS |
| Subject Category: Biochemistry & Molecular Biology; Biophysics |
| IDS Number: LM604 |
| ISSN: 0006-3002 |
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