Mastoparan inhibited PH]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321Nl human astrocytoma cells. Mastoparan inhibited GTPyS-induced, but not Caz+-induced, PH]ino sitol phosphate accumulation in membrane preparations with an K,, of approximately IO pM. The inhibitory effect of mastoparan on carbachol-induced fH]inositol phosphate a~umulation was resistant to pertussis toxin (IAP) reagent in intact cells. These results suggest that mastoparan inhibits phospholipa~ C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.

Yeast peroxisomes were purified to near homogeneity from cells of Candida tropicalis grown on oleic acid for the purpose of examining the possible presence of DNA in this organelle. The purification procedure includes the effective conversion of cells to spheroplasts with Zymolyase and sodium sulfite and the separation of the organelles at extremely low ionic strength. The mitochondrial contamination was less than 1%, based on several criteria, and the yield of peroxisomes was about 40%. The purified peroxisomal fraction contained a very small amount of DNA, which yielded restriction fragments indistinguishable from those of mitochondrial DNA. The absence of DNA in peroxisomes was also supported by cesium chloride density gradient centrifugation of the organelles lysed with a detergent, staining of the organelles with a fluorescent dye specific to DNA, and labeling of the DNA with [3H]adenine.

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