Abstract: The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2‐dipalmitoyl‐ sn ‐glycerol‐3‐phosphoryl[ 3 H]choline and 1‐palmitoyl‐2‐[9, 10(n)‐ 3 H]palmitoyl‐ sn ‐glycerol‐3‐phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X‐100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 m M , but zinc was greatly inhibitory, causing a loss of >90% activity at the 4 m M concentration. Nonhydrolyzable guanine nucleotide analogues such as guanosine 5′‐(3‐ O ‐thio)triphosphate and guanyl‐5′‐yl‐( β,γ ‐methylene)diphosphonate but not guanosine 5′‐(2‐thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5′‐(2‐thio)diphosphate was capable of partially blocking guanosine 5′‐(3‐ O ‐thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was also able to cause a two‐ to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet‐activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide‐binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.
A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.
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