Harris W R, Malencik D A, Johnson C M, Carr S A, Roberts G D, Byles C A, Anderson S R, Heilmeyer L M, Fischer E H, Crabb J W
Protein Chemistry Facility, W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
J Biol Chem. 1990 Jul 15;265(20):11740-5.
A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.
通过有限的胰凝乳蛋白酶消化产生的兔肌肉磷酸化酶激酶的催化片段制剂被分离出来，并通过埃德曼降解法鉴定为γ亚基的NH2末端区域。对活性片段羧基末端肽段的质谱分析、气相序列分析和氨基酸分析表明，在γ亚基序列中的Tyr290、Arg296和Phe298残基处产生了多个COOH末端。这些活性片段种类比γ亚基（Mr 44,673）小约24%，大小范围从Mr 33,279到Mr 34,275。活性片段制剂的比活性比γ亚基-钙调蛋白复合物高约6倍。钙调蛋白赋予γ亚基钙敏感性，但对活性片段的酶学性质没有影响。亲和力测量表明，活性片段与丹磺酰钙调蛋白结合的解离常数为0.7 microM，该值比报道的γ亚基的值弱约28倍。这些数据支持在γ亚基的COOH末端区域存在钙调蛋白结合结构域。
