A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.

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, ...

Concepts and criteria that have been developed for the study of the molecular organization of membrane-associated proteins are employed here to investigate the interaction of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with other membrane components. Detergent-solubilized adenylate cyclase can be shown to bind to erythrocyte-derived Triton X-100 shells containing cytoskeletal elements. This binding appears to be saturable with respect to adenylate cyclase concentration, and it is enhanced by the presence of divalent cations. Preactivation of the enzyme with 5'-guanylyl imidodiphosphate and isoproterenol, or with NaF, is a prerequisite for effective binding. Two exceptions to this general observation are noted: rat brain adenylate cyclase, which binds without prestimulation, and rat testicular cytosolic adenylate cyclase, which fails to bind under any of the conditions tried. The binding sites on the Triton X-100 shells are inactivated or released by treatment with various concentrations of trypsin or KCl. Moreover, exposure of the Triton X-100 shells to increasing temperatures results in a progressive loss of the adenylate cyclase binding capacity. On the basis ofthese and other findings, it is suggested that the adenylate cyclase complex possesses two principal domains that allow it to interact with both cytoskeletal elements and the lipid bilayer. The specific modulation of these interactions may be involved in the hormonal regulation of adenylate cyclase activity.An association between components of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system and the cytoskeleton has been postulated recently (1). Supporting evidence was obtained from the differential solubilization ofthe enzyme from rat erythrocyte ghosts and pigeon erythrocyte plasma membranes with Triton X-100 or low ionic strength buffers (1). Moreover, the effects of cytochalasin B, colchicine, and vinblastine on intracellular levels of 3',5'-cyclic AMP (cAMP) indicate that microfilaments and microtubules may influence the hormonal or cholera toxin stimulation of adenylate cyclase in whole cells (2-4). The morphological co-alignment (5, 6) and the biochemical coprecipitation (7, 8) of actin and certain crosslinked surface antigens suggests a more general role for cytoskeletal components in transmembrane signaling.Not only do membrane proteins that are linked to the cytoskeleton resist solubilization by nonionic detergents but also they can be separated from and reconstituted with other cytoskeletal components. This approach has been employed to investigate the cytoskeletal interactions of band 3 [a transmembrane erythrocyte protein (9)], spectrin [a peripheral membrane protein (10, 11)], and ankyrin [which bridges these two types of proteins (12) Here we have adapted similar techniques to study the binding of adenylate cyclase to the rat erythrocyte cytoskeleton. Our results demonstrate that detergent-solubilized adenylate cyclase binds in a saturable and specific manner to the shells der...

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