One of the major proteins of the Escherichia coli outer cell envelope membrane, protein I, can be separated electrophoretically into protein components la and lb. Strain differences exist regarding presence or absence of component lb and this component can selectively be lost by mutation to resistance against a phage. Both components Ia and Ib are further heterogeneous isoelectrically, and both together may contain at least six separable isoelectric species. As judged by analysis of their cyanogen bromide fragments, la and Ib are almost identical concerning their primary structure; the difference (charge only or size and charge) was located in a part of the protein that does not correspond to the C‐terminal or N‐terminal regions. Components la and Ib thus represent essentially the same polypeptide and they may arise by a modification process in vitro or the existence of two almost identical genes.

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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, ...

Various vasoactive agents (e.g., thrombin and bradykinin) and serum stimulate arachidonate production and thus prostaglandin biosynthesis in cultured fibroblasts. Treatment of 3T3 cells with the anti-inflammatory steroid, dexamethasone, inhibits this stimulation but has no inhibitory effect on the basal activity of phospholipase (or on prostaglandin content) in resting, confluent fibroblasts. In intact cells, the proportion of released arachidonic acid converted into prostaglandins is increased by steroid treatment; in quiescent, dense cells and in serum-treated cells, the total incorporation into prostaglandins is increased. Furthermore, the cyclo-oxygenase activity of homogenates from steroid-treated cells is increased very substantially. Thus, although steroids may affect phospholipase (EC 3.1.1.4),activities it is possible that these effects may be secondary to a more important stimulatory effect on cyclo-oxygenase activity which leads to selective alterations in prostaglandin biosynthesis. The steroid-induced increase in cyclo-oxygenase activity is not observed in a transformed variant of the same cell line. Fatty acid lipoxygenase (EC 1.13.11.12) activity exists in the particulate rather than the cytosolic fraction of 3T3 cells.Prostaglandins E2 and F2a (PGE2 and PGF2a), which are produced by various fibroblast lines in culture (1, 2), have been implicated frequently in inflammatory reactions (3). Although anti-inflammatory steroids have been reported to inhibit prostaglandin synthesis (4), the mechanism of action of steroids has not been clearly established. High concentrations of antiinflammatory steroids have been found to inhibit in ittro preparations of prostaglandin synthetase (5), while other authors observed no inhibition at moderate concentrations (6, 7). Chang et al. (8) reported that steroids prevent the release of prostaglandins rather than affect their biosynthesis. While Gryglewski et al. (9) suggested that steroids reduce the availability of substrate for prostaglandin synthesis, Hong and Levine (10), working with transformed mouse fibroblasts, observed that anti-inflammatory steroids could inhibit phospholipase activity in treated cells and postulated that this inhibition may be fundamental to the anti-inflammatory action of steroid hormones.We have confirmed the inhibitory action of corticosteroids on the stimulated activity of phospholipases (EC 3.1.1.4) in Swiss 3T3 mouse fibroblasts, although the basal enzyme activity is apparently not affected. It is not clear whether this inhibition is a secondary, compensatory response of the intact cell since steroids rapidly induce an increase in fatty acid cyclo-oxygenase activity, the first step in the multienzyme complex known as prostaglandin synthetase.MATERIALS AND METHODS 3T3 (Swiss) fibroblasts and their transformed derivative (SV40, 3T3) were purchased from Microbiological Associates and were cultured in Dulbecco's modified Eagle's minimal essential medium with 10% fetal calf serum, penicillin, and streptomycin. [1-14C]Arachidona...

(5,6). Examples exist where this modulation seems to reflect changes in membrane fluidity (7,8) and others where there appears to be a requirement for certain specific phospholipids for enzyme activity (9-11). Such investigations have been extended to the study of adenylate cyclase principally by the direct addition of phospholipids' (or cholesterol) to cells or membranes by lipic4 fusion or exchange or by supplemention of the medium of auxotrophic mutant cells with phospholipid precursors (12)(13)(14)(15). Although these studies have yielded much new and interesting information, the added lipids may be exerting uncontrolled effects on the metabolism of the cells or they may not partition equally throughout the membrane.We have reported the solubilization of adenylate cyclase from rat brain and its subsequent incorporation into liposomes of defined phospholipid composition (16). These-studies showed that the enzyme activity in the liposomes was dependent on the particular phospholipids present. One drawback to this study is the use of nonionic detergents which, themselves, are capable of stimulating activity. In the present investigation we have used sodium deoxycholate to solubilize the enzyme; this preparation is totally dependent on addition of certain specific phospholipids or nonionic detergent for activity. The results further support the hypothesis that the activity of adenylate cyclase may be dependent on the presence of specific phospholipids.MATERIALS AND METHODS Materials. [a-32P]ATP and cyclic [2,8-3H]AMP were from New England Nuclear; alumina, activity 1, was from ICN. Triton X-100 was obtained from Packard. Pyruvate kinase was purchased from Boehringer Mannheim. Phosphatidyl-N-methylethanolamine (no. 835-8125) and phosphatidylglycerol (no. 835-8126) were supplied by GIBCO. The following naturally derived lipids were from Sigma: phosphatidylcholine (P 5763), lysophosphatidylcholine (L 4129), sphingomyelin (S 7004), phosphatidylserine (P 6641), phosphatidic acid (P 9511), phosphatidylinositol (P 0639), phosphatidylethanolamine (P 4513), phosphatidyl-N,N-dimethylethanolamine (P 1634), cholesterol (CH-S), cholesterol acetate (CH-SA), cholesterol stearate (CH-SS), and cholesterol oleate (CH-SO). ATP, phosphoenolpyruvate, and sodium deoxycholate were also obtained from Sigma. Triolein and 1,3-diolein were purchased from P-L Biochemicals.Preparation of Solubilized Adenylate Cyclase; Male rats (Sprague-Dawley, 120-170 g) were decapitated and their brains were removed. Subsequent operations were performed at 0°C. Each brain was homogenized in 8 vol (vol/wt) of 3 mM MgCl2/ 3 mM dithiothreitol/50 mM Tris-HCl, pH 8.2, and centrifuged for 10 min at 40,000 X gm. This procedure was repeated once and the pellet was then homogenized in 8 vol of 1 mM MgCl2/ 3 mM dithiothreitol/0.5% deoxycholate/50 mM Tris HCl, pH 8.2. The homogenate was incubated at 0°C for 20 min and centrifuged for 40 min at 300,000 x gm.; further centrifugation of the supernatant for 45 min at 300,000 X gm. did not sediment any further enzyme ac...