Kanda Yoshikazu, Hisayasu Sanae, Abe Yasuko, Katsura Kenichiro, Mashimo Keico
Department of Biochemistry, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan.
Life Sci. 2007 Jul 19;81(6):449-57. doi: 10.1016/j.lfs.2007.06.005. Epub 2007 Jun 27.
We determined the growth-active domains of milk-growth factor (MGF), human alpha-lactalbumin (HMLA) and human lysozyme (HMLZ), and their sequences. Fetal calf serum (FCS) showed inhibitors against proteases. The growth-stimulation of IMR90 cells in CG medium (free-serum) without FCS was induced in a dose-dependent manner up to 400 ng/ml of HMLA, HMLZ or chicken lysozyme (ChLZ), and also in a time-dependent manner until 48 h but was induced gradually until 1000 ng/ml of bovine alpha-lactalbumin (BVLA). The HMLAL6-peptide (HMLAL6), a cleaved product from HMLA by Endpeptidase Lys C, was growth-stimulative. The sequence of HMLAL6 was matched to 35 amino-acid residues (from No. 59 to No. 93 of HMLA), owing to the sequences of HMLAL6R3, HMLAL6R5 and HMLAL6R7 after the reduction of HMLAL6. The sequences of the reduced peptides from MGFL7-peptide (MGFL7: a cleaved product from MGF by Endpeptidase lysine C matched to those of the peptides from HMLAL6, and were similarly identified as the partial sequence of HMLA (59-93, H(2)N-L.W.C.?.K./S.S.Q.V.P.Q.S.R.N.I.?.D.I.S.?.D.K./F.L. D.D.D.I.T.D.D.I.M.?.A.-COOH). The sequence of HMLZ is similar to that of HMLA. HMLZT7-peptide (HMLZT7), a cleaved product of HMLZ by trypsin, was confirmed to have growth-stimulating activity and it's sequence was partially identified as Y. W.?.N.D.G.K.T.P.G.A.V.N.A.?.H.L. -, owing to the results of HMLZT7R1 (reduction of HMLZT7) and HMLZA7R2 (reduction of HMLZA7-peptide (HMLZA7) cleaved product of HMLZ by Endpeptidase Arg C) and is accordingly the sequence from No. 63 to No. 97 of HMLZ. Therefore, the peptides produced from LA and LZ by proteolysis may play a role of growth-stimulation.
我们确定了乳生长因子(MGF)、人α-乳白蛋白(HMLA)和人溶菌酶(HMLZ)的生长活性结构域及其序列。胎牛血清(FCS)显示出蛋白酶抑制剂。在不含FCS的CG培养基(无血清)中,IMR90细胞的生长刺激以剂量依赖方式诱导,直至400 ng/ml的HMLA、HMLZ或鸡溶菌酶(ChLZ),并且在48小时内呈时间依赖性诱导,但直至1000 ng/ml的牛α-乳白蛋白(BVLA)时逐渐诱导。HMLAL6肽(HMLAL6)是HMLA经内肽酶Lys C切割产生的产物,具有生长刺激作用。由于HMLAL6还原后的HMLAL6R3、HMLAL6R5和HMLAL6R7序列,HMLAL6的序列与35个氨基酸残基匹配(从HMLA的第59位到第93位)。MGFL7肽(MGFL7:MGF经内肽酶赖氨酸C切割产生的产物)还原后的肽序列与HMLAL6的肽序列匹配,并且同样被鉴定为HMLA的部分序列(59 - 93,H(2)N - L.W.C.?.K./S.S.Q.V.P.Q.S.R.N.I.?.D.I.S.?.D.K./F.L.D.D.D.I.T.D.D.I.M.?.A.-COOH)。HMLZ的序列与HMLA相似。HMLZT7肽(HMLZT7)是HMLZ经胰蛋白酶切割产生的产物,经证实具有生长刺激活性,并且由于HMLZT7R1(HMLZT7还原)和HMLZA7R2(HMLZA7肽(HMLZA7)还原,HMLZA7是HMLZ经内肽酶Arg C切割产生的产物)的结果,其序列部分鉴定为Y.W.?.N.D.G.K.T.P.G.A.V.N.A.?.H.L. -,因此是HMLZ从第63位到第97位的序列。因此,由LA和LZ经蛋白水解产生的肽可能发挥生长刺激作用。