Characterization and partial purification of an insulinase from Neurospora crassa.
An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.
Kole
H K
HK
Department of Physiology and Biophysics, University of Medicine and Dentistry, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Smith
D R
DR
Lenard
J
J
D002413:Cations, Divalent; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007339:Insulysin / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009492:Neurospora crassa / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D007328:Insulin; D013439:Sulfhydryl Reagents; C008147:insulin, iodo-; D004492:Edetic Acid; D007339:Insulysin