Artery membrane fractions

Fung and colleagues examined the metabolic conversion of organic nitrates in sub-cellular fractions of bovine coronary artery smooth muscle cells [66, 67]. They found NO-generating capacity to be present in membrane fractions and, with the use of marker enzymes, identified plasma membrane as the primary location. The enzyme involved in bioconversion was not glutathione-S-transferase [68] and differed from those that catalyse activation of organic nitrites [69]. Partial purification [70] established that the molecular sizes of the native enzyme and subunits were approximately 200 kDa and 58 kDa respectively, and that enzymic activity depends on the presence of a free thiol group. 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

Cell fractionation studies in bovine carotid artery first demonstrated that phorbol esters, histamine, angiotensin, and endothelin, but not KCl, induced a translocation of PKC from the cytosol to the membrane fraction (Haller et al., 1990), although the identity of the membranes involved was not determined (Table I). Work with the non-isoform-specific fluorescent PKC probe Bodipy phorbol (Khalil and Morgan,... 

Protein kinase C Bovine carotid artery Cytosolic fraction Membrane fraction Haller et al. (1990)... 

Phenylarsine oxide, an inhibitor of tyrosine phosphatase, caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable tyrosine phosphatase activity (Su and Block 2000). Phenylarsine oxide also caused a decrease in sulphydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulphydryl content and the inhibitory effect of phenylarsine oxide on eNOS activity were prevented by dithio-threitol, a disulphide-reducing agent. 

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