| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
We first examined the expression level of cytochrome P450 (P450) and catalytic properties of brain microsomes of cynomolgus monkeys. Immunobolt analysis clearly indicated the evidence of expression of P450 in brain microsomes of cynomolgus monkeys. 7-Ethoxycoumarin O-deethylation activity was also detected when cynomolgus monkey brain microsomes were used for the enzyme source. From these results, we ascertained the presence of P450 in monkey brain. Next, we extracted mRNA from rhesus monkey and prepared the cDNA library. Although, immunoscreening was performed by using antibodies raised against rat and human P450s, fragment which was encoded the full-length of brain P450 was not obtained. As antigens used for the immunoscreening were not purified from monkey liver microsomes, we tried to purify P450s from hepatic microsomes of monkey. Four forms of P450s were finally purified from hepatic microsomes of untreated cynomolgus monkeys (P450CMLa-d) and characterized. From doguera baboons,
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, P450 BLa which was closely related to P450 CMLa (P450 3A enzyme) was also isolated and characterized. Antibodies raised against the se P450s were used for immunoscreening of monkey brain P450. However, no positive clone was obtained. On the other hand, the nucleotide sequence of a cDNA coding for monkey cytochrome P450(P450)2B has been determined. Using antibody against P450 CMLa which had been purified from hepatic microsomes of untreated cynomolgus monkeys, a cDNA clone with 2,275 bp insert(Mac2B)was isolated from a lambdagt11 cDNA library prepared from hepatic mRNA from an untreated rhesus monkey. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 491 amino acids. The molecular weight calculated from the deduced amino acid sequence was 55,969. The N-terminal 34 amino acids encoded by Mac2B are identical to those determined by Edman degradation analysis of purified cynomolgus monkey P450 CMLa. The nucleotide and the deduced amino acid sequence of Mac2B were most similar to those of human P450 2B6 among P450 2B subfamily and those sequences of Mac2B were 94% and 90% identical to those of human P450 2B6, respectively. It was also demonstrated that mRNA closely related to Mac2B was detected by Northern blot analysis in rhesus monkey brain. We still continue the sequence analysis for the P450 2B in rhesus monkey brain. Less
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