Major rat brain membrane-associated and cytosolic enkephalin-degrading aminopeptidases: comparison studies.
The major cytosolic and membrane-associated enkephalin-degrading aminopeptidases were purified in parallel by column chromatography successively on DEAE-cellulose, AH-Sepharose, hydroxylapatite, Sephadex G-200, Affigel Blue, AH-Sepharose, and hydroxylapatite. With the final hydroxylapatite column, the cytosol (S) and the membrane (M) enzymes could each be resolved into two peaks, one eluted with 0.05 M phosphate (SI, MI) and the other with 0.25 M phosphate (SII, MII). The overall purification, with Arg BNA as substrate, for the SI and MI was about 450-fold; for SII and MII, 1,200-fold. The yield for each enzyme was about 2%. the major protein integral units of the four enzymes are similar; they are single polypeptide chains with a molecular weight of 100,000 daltons. Their pH optimum, substrate specificity, and sensitivity to puromycin show that they are similar to lysosomal aminopeptidase (EC 3.4.11.-). The two forms of the cytosol and the membrane enzymes have slightly different kinetic constants. With the inhibitors, SII is more sensitive to proctolin, whereas MII is more sensitive to bestatin and Arg-Phe-Ala. Mn2+ activates SI on Met-enkephalin degradation, but inhibits SII, MI, and MII.
Hui
K S
KS
Neurochemistry Division, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962.
Hui
M P
MP
Lajtha
A
A
D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D004745:Enkephalins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D004745:Enkephalins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D008345:Manganese; D000626:Aminopeptidases