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.     

Biosynthesis of various types of collagen by human hepatoma cells in vitro

A cloned human hepatoma cell line (HH2-1) produced and formed collagen fibers in vitro. The relative rate of collagen synthesis by the cells was increased with an enhancement of the cell density. An analysis of the components of the collagen using sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the cells synthesized interstitial collagen, types I and III, and other collagenous proteins. Thus, human hepatoma cells may play an important role in the formation of stromal collagen in the tumor.     

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.     

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.     

Studies on functional domains of the regulatory subunit of bovine heart adenosine 3':5'-monophosphate-dependent protein kinase

The functional domains of the regulatory subunit of isozyme II of cAMP-dependent protein kinase were studied. It was shown using Edman degradation that the regulatory subunit contained a phosphorylated residue which was very close in primary sequence to the site most sensitive to hydrolysis by low trypsin concentrations as postulated previously (Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.M., and McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003). Catalytic subunit incorporated 0.9 mol of 32P from [gamma-32P]ATP into a preparation of regulatory subunit that contained 1.1 mol of endogenous phosphate. After phosphorylation by the catalytic subunit, the regulatory subunit contained 2.2 mol of chemical phosphate. The effects of heat denaturation upon the rate and extent of phosphorylation of the regulatory subunit were compared with the effects of these treatments upon the cAMP binding and inhibitory domains. These data suggested that the regulatory subunit required factors in addition to an intact phosphorylatable primary sequence in order for inhibitory activity to be expressed. Such factors might be part of the secondary or tertiary structure of the protein. These studies are discussed with respect to the mechanism of inhibition of catalytic activity, and a model of the regulatory subunit structure is proposed.