The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process that produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.The molecular mechanisms of activation of adenylate cyclase by NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p], and the relevance of such mechanisms to the hormonal modulation of this enzyme, have been difficult to investigate due to the lack of appropriate techniques for the resolution and reconstitution of the various molecular components of the adenylate cyclase system. We have reported (1) the development of such a technique which involves the preparation of a Gpp(NH)p-and NaF-insensitive brain particulate adenylate cyclase by differential detergent extraction. The enzyme regained its characteristic Gpp(NH)p and NaF responses upon the addition of one or more proteins solubilized from membrane preparations of various tissues (1). These regulatory proteins can be partially separated from solubilized adenylate cyclase by gel filtration on Ultrogel AcA 34 columns. We report here the utilization of this reconstitution technique to help elucidate the mode of interaction of the various components of this multifactorial system. Thermal and trypsin inactivation of the Gpp(NH)p-and NaF-reconstituting activities suggests that the two activities are functionally separable. Gpp(NH)p and NaF appear to interact reversibly with the corresponding regulatory proteins in a divalent cation-independent step, followed by a divalent cation-dependent step that leads to the persistent state of enzyme activation. Techniques that separate regulatory from catalytic components can also reverse the enzyme activation.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3693 The Gpp(NH)p-and NaF-insensitive adenylate cyclase was prepared by a modification of a published procedure (1, ...

ATP depletion is always associated with prolonged ischemia. It was found that ATP affected calcium- and phospholipid-dependent activation of protein kinase C without hydrolysis of the nucleotide when the activation was monitored by an assay for [3H] 4-beta-phorbol-12, 13-dibutyrate binding activity in a reconstitution system having physiological concentrations of free calcium. When the ATP level was low, an increase in the free calcium concentration could not activate the enzyme. A decrease in pH exacerbated the depressed activation. The concentration of magnesium also affected the activation. On the other hand, free fatty acids, which increase during ischemia, were able to activate the enzyme at a low concentration of ATP in the absence of phorbol ester and phosphatidylserine. These results suggest that calcium- and phospholipid-dependent activation of protein kinase C is suppressed during ischemia, and that fatty acids in turn activate the enzyme. It is possible that ischemia interferes with normal signal transduction via the protein kinase C pathway and causes unusual protein phosphorylation.

Treatment of human peripheral blood lymphocytes (PBL) with phorbol dibutyrate (PDBU) for 20 to 45 min at 37 degrees C induces adherence of 5 to 30% of the cells to plastic. The adherent cells (pAd) were highly enriched in NK cells on the basis of the following findings: 1) they exhibit high NK and ADCC activity but do not lyse the NK-resistant cell line, Daudi; 2) cytotoxic activity is enhanced by pretreatment with interferon-alpha (IFN-alpha); 3) the surface markers of these cells, as determined with monoclonal antibodies, are consistent with NK cells; and 4) they are enriched with cells morphologically similar to large granular lymphocytes. Conversely, the PDBU-nonadherent cells were substantially depleted of NK cells. The fact that the pAd cells do not lyse the Daudi line and that their NK activity can be further augmented by IFN-alpha would suggest that the pAd are enriched for NK cells rather than changed in their characteristics as a result of the separation procedure. Moreover, no consistent and appreciable modulation of NK activity induced by PDBU was observed. This report therefore demonstrates that PDBU selectively induces NK adherence of NK cells, which may have practical as well as biological implications.