Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

A 70-kDa protein is phosphorylated in cell-free preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the 70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol of normal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed.

Stimulation of 32Pi transport into human erythrocyte ghosts and reconstituted vesicles by Mg2+ and hemoglobin.

Human erythrocyte ghosts catalyze a low rate of 32Pi uptake. A severalfold stimulation of 32Pi uptake was observed after exposure of the membranes to an erythrocyte lysate or to hemoglobin in the presence of Mg2+. Ghosts prepared from erythrocytes that had been exposed to 10 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked reduction in 32Pi uptake. Reconstitution of membranes with added phospholipids by freezing and thawing, by octylglucoside dilution or by cholate dialysis, yielded vesicles that catalyzed 32Pi uptake. When membranes were incubated with hemoglobin and Mg2+ prior to reconstitution, the rate of uptake was increased severalfold. The inhibition of hemoglobin and Mg2+-dependent uptake of 32Pi by chloride suggests that the transport in the reconstituted vesicles is catalyzed by the classical inorganic anion transporter.

Acetyl phosphatidylethanolamine in the reconstitution of ion pumps.

Acetyl phosphatidylethanolamine was compared with phosphatidylethanolamine in the reconstitution of several biological membrane activities with the following results. 1. The proton pump reconstituted with the purple membrane of Halobacterium halobium and acetyl phosphatidylethanolamine was quite active. However, some differences in the kinetic properties, particularly in the decay rate, were noted between vesicles reconsituted with phosphatidylethanolamine and acetyl phosphatidylethanolamine. 2. Acetyl phosphatidylethanolamine could not replace phosphatidylethanolamine in the reconstitution of a Ca-2 plus pump with ATPase isolated from sacoplasmic reticulum. However, inclusion of suitable amounts of stearylamine or oleylamine during reconstitution yielded acetyl phosphatidylethanolamine vesicles with Ca-2 plus translocation activity comparable to that of phosphatidylethanolamine vesicles. 3. A mixture of acetyl phosphatidylethanolamine and stearylamine or oleylamine substituted for phosphatidylethanolamine in the reconstitution of mitochondrial hydrophobic proteins to form vesicles that catalyze 32-Pi-ATP exchange. Since phosphatidylcholine is also required in this system, these findings point to two functions of phosphatidylethanolamine, one related to the specific properties of its amino group, the other to a structural role of its small polar head group. A hydrophobic alkylamine can fullfill the first function, acetyl phosphatidylethanolamine the second. 4. The importance of the charge was also observed in experiments with the reconstituted rutamycin-sensitive ATPase of mitochondria. After depletion of phospholipids from the hydrophobic proteins, ATPase activity and rutamycin sensitivity were restored only if a phospholipid as well as the appropriate charge were present.