Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

Enhancement of eosinophil effector function by soluble factors released by S. mansoni: role of proteases

Schistosomulum-released products (SRP) have been shown to enhance both expression of rat and human eosinophil Fc receptors and IgG-dependent cytotoxicity. The present work provides additional evidence of the secretion of eosinophil-enhancing factors by schistosomula and other developmental stages of schistosomes, including adult worms. The heat lability, as well as the strong inhibition of the stimulating activity of SRP by the protease inhibitor Trasylol, suggest that thermolabile proteases secreted by the parasite are involved in this mechanism. The purification of the schistosome proteases by preparative isoelectric focusing and gel filtration demonstrated that neutral proteases able to hydrolyze the collagenase substrates Azocoll and Z-Gly-Pro-Leu-Gly-Pro are able to significantly enhance eosinophil effector functions. Purified Clostridium histolyticum collagenase was also able to mimic the enhancing effect of schistosome proteases, suggesting involvement of a collagenase-like activity of the enzymes in the eosinophil stimulation.     

Regulation of ATP synthesis catalyzed by the calcium pump of sarcoplasmic reticulum

The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum.     

Tryptic cleavage and substructure of bovine cardiac myosin subfragment 1

The method of limited tryptic proteolysis has been used to compare and contrast the substructure of bovine cardiac myosin subfragment 1 (S-1) to that of skeletal myosin S-1. While tryptic cleavage of cardiac S-1, like that of skeletal S-1, yields three fragments, the 25K, 50K, and 20K peptides, the digestion of cardiac S-1 proceeds at a 2-fold faster rate. The increased rate of cleavage is due entirely to an order of magnitude faster rate of cleavage at the 25K/50K junction of cardiac S-1 compared to that of skeletal, with approximately equal rates of cleavage at the 50K/20K junctions. Actin inhibits the tryptic attack at this latter junction, but its effect is an order of magnitude smaller for the cardiac than for the skeletal S-1. Furthermore, the tryptic susceptibility of the 50K/20K junction of cardiac S-1 in the acto-S-1 complex is increased in the presence of 2 mM MgADP. This effect is not due to partial dissociation of the cardiac acto-S-1 complex by MgADP. Our results indicate that in analogy to skeletal S-1, the cardiac myosin head is organized into three protease-resistant fragments connected by open linker peptides. However, the much faster rate of tryptic cleavage of the 25K/50K junction and also the greater accessibility of the 50K/20K junction in the cardiac acto-S-1 complex indicate substructural differences between cardiac and skeletal S-1.