Simple preparation of rat brain lysosomes and their proteolytic properties.
Brain lysosomes were isolated from rat cerebra by Percoll density gradient centrifugation. The lysosomes had little and no contamination by marker enzymes from mitochondria and other organellae, respectively, and the yield was approximately 14% of the postnuclear supernatant. The activities of cathespins B, L, and/or J were similar to those of liver or kidney lysosomes, but the levels of cathepsin H activity were much lower than those of liver or kidney lysosomes. The degradation of native L-lactate dehydrogenase (LDH) and rat serum albumin by the isolated brain lysosomes in vitro was markedly suppressed by a low level of the cysteine proteinase inhibitor cystatin alpha, with slight inhibition of the activities of cathepsins B, L, and/or J. The degradation of rat serum albumin was also considerably inhibited by N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)- L-isoleucyl-L-proline (CA-074), a selective inhibitor of cathepsin B. In contrast, the degradation of brain proteins from the postmitochondrial supernatant by the same brain lysosomes was not or little suppressed by the same concentration of either inhibitor. However, it was considerably suppressed by leupeptin with marked inhibition of the activities of cathepsins B, L, and/or J, and with only slight inhibition of cathepsin H, indicating that cysteine proteinases that are highly sensitive to leupeptin are involved in the lysosomal degradation of the brain proteins. It was also moderately suppressed by pepstatin, an inhibitor of cathepsin D and was almost completely suppressed by a combination of leupeptin and pepstatin.(ABSTRACT TRUNCATED AT 250 WORDS)
Ohshita
T
T
Division of Enzyme Chemistry, University of Tokushima, Japan.
Kido
H
H
D000818:Animals; D001192:Arylsulfatases / Q000737:chemistry; D001921:Brain / Q000648:ultrastructure*; D002403:Cathepsins / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D003102:Colloids; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011205:Povidone; D011480:Protease Inhibitors / Q000737:chemistry; D051381:Rats; D012822:Silicon Dioxide; D013385:Succinate Dehydrogenase / Q000737:chemistry
D003102:Colloids; D011480:Protease Inhibitors; C016039:Percoll; D012822:Silicon Dioxide; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D001192:Arylsulfatases; D002403:Cathepsins; D010447:Peptide Hydrolases; D011205:Povidone