Analyses on sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that the human hair cuticle extracts mainly consist of a 7-kDa component and keratin proteins. The S-carboxymethylation of the cuticle extracts made the 7-kDa band shift to the 15-kDa position. After electroblotting of the S-carboxymethyl derivative, the membrane pieces carrying the 15-kDa band were treated with trypsin and the released peptides were separated by reverse-phased HPLC. Amino acid sequence analyses revealed that the peptides corresponded to the partial sequences deduced from human genome coding for S100A3, a cysteine-rich calcium binding protein. The anti S100A3 serum, prepared by immunizing a synthetic peptide antigen, reacted with the 7-kDa and 15-kDa bands in immunoblotting analyses. Immunofluorescence microscopy showed intense labeling to the cuticular layer with the anti S100A3 serum. These results indicated that S100A3 was highly expressed in the human hair cuticle.
The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.
SUMMARYPeptides that inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase were isolated from a pepsin digest of α-casein. Analysis of these peptides showed that they corresponded to the αs2-casein sequences 164–179 (Leu–Lys–Lys–Ile–Ser–Gln–Arg–Tyr–Gln–Lys–Phe–Ala–Leu–Pro–Gln–Tyr), 183–206 (Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr) and 183–207 (C-terminus, Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr–Leu). These peptides inhibited calmodulin-induced cyclic nucleotide phosphodiesterase activity over the range 1–50 μM without affecting the basal enzyme activity. These results demonstrated that the affinities of these peptides for calmodulin are comparable to the affinities of certain endogenous neurohormones and proteins that interact with calmodulin.
A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropomyosin, troponin, the 150,000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel electrophoresis of the purified myosin showed three closely spaced bands corresponding to the 20,000 dalton, the charge-modified 20,000 dalton and the phosphorylated 20,000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55 muM and the Vmax values were 0.263 and 0.317 mumole P1/mg/min for the Ca2+-activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.
Calmodulin-binding peptides, which had previously been isolated from a pepsin digest of alpha-CN, were synthesized and then examined for their inhibitory effects on the activation of cyclic nucleotide phosphodiesterase that was induced by calmodulin. The concentrations of the synthetic peptides corresponding to 164-179, LKKISQRYQKFALPQY; 183-206, VYQHQKAMKPWIQPKTKVIPYVRY; and 183-207, VYQHQKAMKPWIQPKTKVIPYVRYL, of alpha s2-CN that gave half-maximal inhibition were 65, 7.0, and 2.6 microM, respectively. These inhibitory effects were reversed by increasing the amount of calmodulin. Fragments and analogs were prepared to study the interactions of the peptides with calmodulin in more detail. The results indicated that modification of the carboxyl terminus enhanced the affinities of the three peptides for calmodulin, and a region involved in the inhibition by alpha s2-CN (f183-207) was located at the carboxyl terminus 191-207. Two predicted calmodulin-binding sequences, 164-179 and 191-207 of alpha s2-CN, despite rather divergent primary structures, shared the structural motif common to the calmodulin-binding domains of the target proteins in the previously proposed complex model.
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