Establishment of highly specific radioimmunoassays for neurokinin A and neurokinin B and determination of tissue distribution of these peptides in rat central nervous system.
Highly specific radioimmunoassays (RIAs) for neurokinin A (NKA) and neurokinin B (NKB) were developed. Antisera were produced by the procedure which involved immunization with NKA or NKB, both conjugated with keyhole limpet hemocyanin, and treatments with a tolerogenic conjugate of kassinin and a copolymer of D-glutamic acid and D-lysine (D-GL) to inhibit the production of cross-reactive antibodies against common C-terminal region of tachykinins. Cross-reactivities of anti-NKA antiserum (R704), thus produced, with NKB, kassinin, eledoisin were 12.6%, 10.6% and 11.5%, respectively. This was in sharp contrast with those of antiserum obtained from the rabbit not treated with kassinin-D-GL, these values corresponding to 129.0%, 42.5% and 94.4%, respectively. The cross-reactivities of R704 with substance P and physalaemin were 0.3% and 1.5%, respectively. This antiserum also bound 35.6% of neuropeptide K which contains NKA at its C-terminal. More importantly, anti-NKB antiserum (R707) obtained by the above tolerizing regimen was highly specific for NKB and the cross-reactivities with NKA, neuropeptide K, kassinin and other tachykinins were all less than 0.001%. RIAs using these specific antisera allowed us to measure directly NKA and NKB in tissue extracts without their fractionation by chromatography prior to RIAs. Measurements of immunoreactive NKA and NKB in different rat brain regions and spinal cord revealed that they are present with various ratios (NKA/NKB: 1.1-9.9) depending on the region.
Tateishi
K
K
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Matsuoka
Y
Y
Hamaoka
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D003429:Cross Reactions; D007106:Immune Sera; D008297:Male; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000032:analysis*; D015287:Neurokinin B / Q000032:analysis*; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values; D013116:Spinal Cord / Q000032:analysis*
D007106:Immune Sera; D015288:Neurokinin A; D015287:Neurokinin B