ABSTRACT
Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.
Subject(s)
Aspartic Acid Endopeptidases/chemistry , Cell Membrane/enzymology , Golgi Apparatus/enzymology , Isoenzymes/chemistry , Metalloendopeptidases/chemistry , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/analysis , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Base Sequence , CHO Cells , Cell Line , Cloning, Molecular , Cricetinae , Endothelin-1 , Endothelin-Converting Enzymes , Endothelins/metabolism , Fluorescent Antibody Technique , Humans , Isoenzymes/analysis , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Metalloendopeptidases/analysis , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Protein Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleases/metabolism , Sequence Analysis, DNAABSTRACT
A description of a lightweight small pupil binocular indirect ophthalmoscope is presented. This instrument can be used with well dilated pupils and clear media as any stereoscopic indirect ophthalmoscope. However, the instrument can be simply and easily adjusted for usage in patients with small pupils or opacities of the media. The optical and mechanical principles employed are presented. The technique of utilization for small pupil viewing is outlined.
