The phorbol diester receptor present in the particulate fraction of rat brain was solubilized by divalent ion chelation in the absence of detergents. The soluble receptor was partially purified by (NH4)2SO4 precipitation, DEAE-cellulose, and gel filtration chromatography. The purified receptor required exogenous phospholipid for activity and displayed a Kd of7 nM for[H]phorbol 12,13-dibutyrate. Biologically active phorbol analogs inhibited binding, whereas inactive analogs did not. The Ca2+-dependent, phospholipid-sensitive protein Iinase C copurified with the phorbol receptor. Purified protein kinase C was activated directly by phorbol 12-myristate 13-acetate in the presence of phospholipid.The tumor-promoting phorbol diesters are agents that are noncarcinogenic but will induce tumor formation when administered after subthreshold doses ofa carcinogen (1). In an attempt to understand the mechanism of tumor promotion, the phorbol diesters have been studied in many systems and have been shown to induce a myriad ofin vitro functional and biochemical changes. These changes, which may be related to cocarcinogen activity, include morphological transformation (2), enhanced hexose transport (3), enhanced prostaglandin synthesis (4), altered phospholipid and protein synthesis (5, 6), loss of fibronectin (7), increased ornithine decarboxylase activity and polyamine production (8, 9), and altered rates ofDNA synthesis (10-13). The phorbols also induce secretion (14-15), superoxide production (16), and alteration of surface receptors (17)(18)(19) and influence differentiation of cells in vitro; most notably, all human myeloid leukemia cells can be induced to differentiate into macrophage-like cells (20)(21)(22). For induction of many of these responses, similar structure-function relationships were shown for a series of phorbol diester analogs, suggesting that each of these diverse responses was mediated via a common pathway.Further support for a common pathway has come from studies demonstrating high affinity, saturable, stereospecific receptors that mediate the activity of the phorbol diesters in many different cell types (23)(24)(25)(26)(27)(28). Although the binding properties of these receptors have been characterized extensively, their biochemical structure or possible mechanism of transduction is unknown.Recently, Castagna et aL were able to show direct activation of partially purified protein kinase C by active phorbol diester analogs (29). Based on these observations, they put forth the exciting hypothesis that protein kinase C may be a membrane target of the phorbol diesters. In this manuscript we present data that extends this hypothesis. The phorbol diester receptor of rat brain membranes, solubilized by divalent ion chelation in the absence of detergents, copurifies with protein kinase C.In the latter steps of purification, both activities require added phospholipid.MATERIALS Sprague-Dawley female weanling rats were a gift from P. M. Conn; Sephacryl S-200 was from Pharmacia (Uppsala, Sweden); DE 52 was...

Tetramethylrhodamine labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys is a potent chemoattractant for human neutrophils. Binding of this peptide to living neutrophils was observed by means of video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the fluorescent peptide. These processes are dependent on specific binding to the formal peptide chemotactic receptor.

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