Rat brain cathepsin L: characterization and differentiation from cathepsin B utilizing opioid peptides.
The specificity of purified rat brain cathepsin L (EC 3.4.22.15) was mapped by the use of synthetic and opioid peptides and some properties were compared to rat brain cathepsin B, rat kidney cathepsin L, and bovine spleen cathepsin C. Brain and kidney cathepsin L cleaved leucine or methionine enkephalin (LE or ME) at the Gly-Gly bond to release Tyr-Gly and Gly-Phe-Leu (-Met). In studies on pro-opioids, the brain enzyme also recognized Met-Arg, Arg-Arg, and Arg-Ile bonds; the best substrates on a relative basis were ME-Arg-Phe, LE- or ME-Arg-Arg, and LE-Arg-Arg-Ile. Measurement of kinetic values in relation to the sites of opioid cleavage provided a basis to differentiate brain cathepsins B and L. Cathepsin L acted with high affinity toward LE to cleave Gly2-Gly3 (Km 82.5 microM, kcat 2034 min-1), in contrast to low affinity cleavage by cathepsin B at Gly3-Phe4. Kapp, the second-order rate constant of enzyme inactivation by Z-Phe-Phe-CHN2 with LE as substrate was 31,530 M-1 s-1 or 10(3) higher than its effect on cathepsin B-mediated hydrolysis of ME-Arg-Phe at the Met-Arg site. Gly-Gly cleavage by cathepsin L was blocked by D-Ala2, did not require the presence of free end groups, and was the only site recognized within opioid peptides having a C-terminal Arg-COOH. The use of opioid peptides as substrates provides further insight into cathepsin L specificity. For these the susceptible sites were flanked primarily by hydrophobic and aromatic groups at P2, P2' or P3'.
Center for Neurochemistry, Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, New York 10035.
N
N
Marks
M J
MJ
Berg
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002401:Cathepsin B / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004723:Endorphins / Q000378:metabolism*; D004743:Enkephalin, Leucine / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D051381:Rats; D013379:Substrate Specificity
D004723:Endorphins; D004743:Enkephalin, Leucine; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; C534280:Ctsl protein, rat