OKAMOTO Hideaki Saga Medical School, Dept. of Pharmacology, Research Assistant, 医学部, 助手 (70315183)
TAKANO Masako Saga Medical School, Dept. of Pharmacology, Research Assistant, 医学部, 助手 (00154807)
|Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
1) Novel ATP/DTT-Stimulated GC; ATP/DTT-stimulated guanylate cyclass (GC) in lung membrane was stimulated 18-fold by ATP and DTT, and both its activity and ANP-stimulated GC activity were observed to be additive. ATP/DTT-stimulated GC was solubilized by octyl glucoside (OG) to examine the mechanism of ATP/DTT-stimulation. GC in OG-extracts was stimulated maximally 2.5-fold by both ATP, ATPγS or AMPPNP, and DTT. Preincubation of OG-extracts at 10ーC with AMPPNP and DTT (Ist-preincubation) converted GC to an insensitive state to stimulation by both ATP and DTT, and this conversion was partly inhibited by a proteinphosphatase-1 inhibitor (10-1000 nM okadaic acid). On the other hand, ANP-stimulated GC was not converted to an insensitive state to ANP/ATP-stimulation by the 1st-preincubation. And, subsequent preincubation of OG-extracts at 10℃ with both DTT and, ATP or ATPγS but not AMPPNP converted GC to a state sensitive to ATP/DTT-stimulation, and this conversion was partly inhibited by in
hibitors of Ca^<2+>/calmodulin-dependent protein kinase II (KN 62 and KN-93). In contrast, the preincubation with KN-62 and KN-93 had no effect on ANP-stimulated GC activity. The results suggested that phosphorylation was involved in the regulation of ATP/DTT-stimulated GC sensitivity to ATP/DTT-stimulation, and that ATP/DTT-stimulated GC activity was likely to be a different type from ANP-stimulated GC activity.
2) Measurement of Membrane Protein; Accurate determination of low levels of protein in samples containing large amounts of interfering substances is rather difficult. In this report, we described an improved method, the DOC-TCA-washing-BCA method, which enabled us to perform rapid and efficient removal of many interfering substances, and to allow proteins to be detected above 0.05μg.
3) New variant of β2 Subunit of Soluble GC (β2b) ; We isolated two distinct cDNA fragments for the β2 subunit of soluble GC (β2a and β2b) from a rat brain cDNA library. The deduced amino acid sequence of β2b is C-terminally shorter by 46 amino acids and thus does not contain a consensus sequence for isoprenylation / carboxymethylation. RT-PCR analysis demonstrated that both variants are expressed in various tissues, including kidney, liver, and brain. Less