Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells
| Autor: | V C Manganiello, M A Danello, M Vaughan, Gordon J Strewler |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1983 |
| Předmět: |
Male
Calmodulin chemistry.chemical_element Calcium Biochemistry Isozyme Chromatography DEAE-Cellulose Liver Neoplasms Experimental 3' 5'-Cyclic-GMP Phosphodiesterases Animals Chymotrypsin Molecular Biology Cells Cultured chemistry.chemical_classification biology Cyclic nucleotide phosphodiesterase Activator (genetics) Phosphodiesterase Cell Biology Molecular biology Rats Isoenzymes Molecular Weight Kinetics Enzyme chemistry Liver biology.protein Research Article |
| Popis: | We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected. |
| Databáze: | OpenAIRE |
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