DOHI Shuji Gifu University, School of Medicine, Professor, 医学部, 教授 (40155627)
NOZAKI Masakatsu Gifu University, School of Medicine, Associate Professor, 医学部, 助教授 (30021380)
OHTA Shuichiro Gifu University, School of Medicine, Assistant Professor, 医学部, 講師 (50144027)
|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥1,700,000 (Direct Cost : ¥1,700,000)
We have recently developed a high affinity antisera that recognizes the epitope representing the mid-portion of SP,i.e., SP4-10. Anti-SP-47 is probably the mid-portion anti-SP sera, that does not display strong preference for the free NH2-terminus of the molecule.
For the present study, rat dorsal root ganglia (DRG) were extracted in 2N acetic acid, followed by an additional CH3CN.After lyophilization, reconstituted extracts were fractionated by analytical reversed-phase HPLC performed with a Vydac C18 column using 3 consecutive linear gradients of CH3CN in TFA,0.5 ml fractions were collected. Standard retention time were calculated by RIA analyzes performed on aliquots of collected fractions after HPLC fractionation of synthetic SP,SP-G,SP-GK,and SP-G-K-R.
Specific RIAs for SP,SP-G,and SP-G-K were performed on HPLC fractions.
Based on these analyzes, we have initially established intrinsic molar ratios of immunoreactivities corresponding to SP and its graded series of small unamidated precursors, i.e., SP-G-K-R,SP-G-K,and SP-G in rat DRG.The respective RIA values for SP-G-K-R,SP-G-K,SP-G,and SP were 2.5,1.0,5.7, and 86.6% of the total recovered immunoreactivity. Thus, it is highly probable that these small peptide determinants are genuine intermediates in the normal pathway of biosynthetic maturation of SP.