Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.
Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.
Thompson
W J
WJ
Department of Pharmacology, University of South Alabama College of Medicine, Mobile 36688.
Tan
B H
BH
Strada
S J
SJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D005978:Glutathione / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000494:pharmacology*; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D014638:Vanadates; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D005978:Glutathione; D006152:Cyclic GMP