Simple preparation of rat brain lysosomes and their proteolytic properties

Analytical Biochemistry
Takeyuki Ohshita, H Kido

Abstract

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, a...Continue Reading

Citations

Jun 13, 2002·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Momono YoshikawaSei Sasaki
Nov 24, 2007·BMC Neuroscience·Rona WilsonJames Brewer
Mar 15, 2005·Journal of Neuroscience Research·Edward RockensteinEliezer Masliah
May 6, 2005·American Journal of Physiology. Cell Physiology·Ryuji BitoHiroaki Kawabata

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