The pulmonary isozyme of angiotensin-converting enzyme (ACEP) is present in the body both as a cell-associated protein in endothelial, epithelial, and monocytic cells and as a soluble protein in various body fluids including serum. The mechanism by which soluble ACEP is produced in vivo is unknown. Using in vitro transfected cell culture systems, we previously demonstrated that the rabbit testicular isozyme of ACE (ACET), which shares extensive homology with ACEP, is first synthesized as a plasma membrane-anchored ectoprotein and then secreted to the culture medium by cleavage removal of its COOH-terminal membrane-anchored tail. Here, using in vitro cultures of arterial endothelial cells and acutely isolated renal epithelial cells, we demonstrate that ACEP is also cleavage secreted from their natural producer cells. Biochemical and immunological characterization of the in vitro secreted ACEP protein revealed that it is missing the COOH-terminal membrane-anchored region of the cell-associated ACEP. Similar analysis of ACEP proteins present in rabbit serum, lung, and kidney established that ACEP secretion in vivo is also caused by the cleavage removal of the COOH-terminal region of the cell-associated protein. To characterize the proteolytic enzyme responsible for ACEP secretion, we employed rabbit renal proximal tubular epithelial cells and demonstrated significant inhibition of secretion by compound 3, a hydroxamic acid-based inhibitor of specific metalloproteases. In contrast, the inhibitors of chymotrypsin, trypsin, serine, aspartate, and cysteine proteases were ineffective. These results indicate that soluble ACEP production by vascular endothelial and renal epithelial cells, both in vitro and in vivo, is achieved by cleavage removal of its membrane-anchoring COOH-terminal tail by a metalloprotease.

Angiotensin-converting enzyme (ACE) is a type I glycoprotein anchored in the plasma membrane by a hydrophobic domain near its carboxyl terminus. The enzymatically active extracellular domain of ACE is slowly released from the cell by cleavage-removal of its membrane-anchoring carboxyl-terminal region. In the present study, we investigated the role of N- and O-glycosylation in intracellular transport and extracellular cleavage-secretion of rabbit testicular ACE. For ACE expression, we used an in vitro translation system, a permanently transfected mouse cell line, and human and Chinese hamster cells transiently transfected with vaccinia virus-T7 RNA polymerase-driven expression vectors. Sugar modifications of ACE were analyzed by testing its sensitivity to specific glycosidases. Cellular protein glycosylation was inhibited by using chemical inhibitors and a mutant cell line defective in protein glycosylation. Our experiments demonstrated that newly synthesized ACE acquires both N- and O-linked sugars before its cleavage-secretion and complete blockage of glycosylation results in rapid intracellular turnover of underglycosylated ACE. However, ACE synthesized without N-linked complex sugars and O-linked sugars can undergo normal transport and cleavage-secretion, and the underglycosylated protein is enzymatically active.

SUMMARYThe M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM),is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing approximately 10% of protein, approximately 30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of beta-actin. Treatment of hypothyroid rats with T(3) increased the relative abundance of both alpha1 and beta1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T(3)-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of alpha1 and beta1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.