Orientation of the brush-border membranal proteinase which specifically splits the catalytic subunit of cAMP-dependent protein kinase.

The active site of the rat intestinal brush-border membranal proteinase [Alhanaty E. and Shaltiel S. (1979) Biochem. Biophys. Res. Commun. 89, 323-332], which splits the catalytic subunit (C) of cAMP-dependent protein kinase with a remarkable specificity [Alhanaty E., Tauber-Finkelstein, M., Schmeeda, H. and Shaltiel, S. (1985) Curr. Topics Cell. Regul. 27, 267-277], is shown to face predominantly the cell exterior; vesicles prepared from these brush-borders (mostly sealed and right-side-out) fully express the proteinase activity as judged by the fact that there is no increase in activity upon rupture or solubilization of the vesicles. Although the brush-border vesicles contain a cAMP-dependent protein kinase, this membrane-bound kinase is not likely to be the physiological target of the proteinase, since it appears to have an intracellular orientation and, at least in the vesicles, to be inaccessible to the proteinase. It is, therefore, suggested that the physiological substrate of the proteinase might be either an extracellular cAMP-dependent protein kinase, which is lost (e.g. removed, inactivated or degraded) in the course of vesicle isolation, or a kinase domain in one of the family of proteins recently shown to have a considerable structural and conformational homology with C. Alternatively the physiological site of action of this kinase-splitting proteinase might be an intracellular organelle to which it is translocated by endocytosis.