Phospholipase microsome preparation

Further support for the hypothesis that Ca2+ plays a central role in regulating phytoalexin accumulation is provided by experiments in which the turnover of phosphatidylinositol was measured in the plasma membrane of elicitor-treated carrot cells [17]. The carrot cells were first labelled with [3H]myo-inositol and, after the addition of elicitors, acid extracts of the cells were analyzed chromatographically for the production of inositol trisphosphate (IP3). In cells treated with elicitor, the release of radioactive IP3 increased with time and attained a maximum at 3 - 5 min after treatment. Phospholipase activity responsible for the degradation of phosphorylated phosphatidylinositol increased correspondingly. Several reports have shown that IP3 induces rapid release of Ca2+ from intracellular stores in animal cells [18, 19]. Studies on plant cells have also demonstrated that exogenous IP3 releases Ca2+ from microsomal preparations at micromolar concentrations, although only limited... 

In 1954, Beaufay and de Duve (27) first suggested a relationship between microsomal phospholipid and glucose-6-phosphatase. They observed a loss of enzymic activity from phospholipid-rich microsomal preparations concomitant with extraction with such organic solvents as butanol or treatment with lecithinase. Various studies were carried out to demonstrate that the latter effect was not produced through inhibition of enzymic activity by accumulated products of the hydrolysis of phospholipids. On the basis of their observations that deoxycholate treatment labilized microsomes to phospholipase action, they concluded that . . . the detergent did not exert its primary effect on the dissociation of phospholipids from microsomal protein, but that it probably disrupted... 

ER of adrenal, testis, ovary, liver and placenta. It is relatively unstable, being inactivated by freezing, even when pure. A phospholipid environment appears to be an important requirement since, when bovine adrenal microsomal preparations were treated with phospholipase A, 80-85% of phospholipids were hydrolysed with a concomitant loss of 80-90% of enzymic activity [84], Restoration of activity was achieved by adding back to the lipid-depleted membranes aqueous dispersions of microsomal total lipid mixtures [84],... 

Na-K ATPase is an intrinsic membrane protein, which requires phospholipids for its activity. Lipid removal from crude microsomal preparations by detergents, organic solvents or phospholipase treatment leads to partial or complete inactivation of the... 

The alcohol once formed is oxidized by an NAD(P)H- and 02-dependent oxygenase to the aldehyde and then to the carboxylic acid. The 4a-carboxylic acid is oxidized by an NAD " -dependent enzyme to the 3-oxo-4a-carboxylic acid. This enzyme has been solubilized and purified from rat liver microsomes. The enzyme has a of 7 juM for the sterol substrate and is maximally active at alkaline pH. The preparation was free of the hydroxylase activities and was uninfluenced by treatment with several phospholipases. It is not known whether the j8-oxo acid formed decarboxylates spontaneously or enzymatically [105]. 

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