The complete amino acid sequence of human von Willebrand factor (vWF) is presented. Most of the sequence was determined by analysis of the S-carboxymethylated protein. Some overlaps not provided by the protein sequence analysis were obtained from the sequence predicted by the nucleotide sequence of a cDNA clone [Sadler, J.E., Shelton-Inloes, B.B., Sorace, J., Harlan, M., Titani, K., & Davie, E.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6391-6398]. The protein is composed of 2050 amino acid residues containing 12 Asn-linked and 10 Thr/Ser-linked oligosaccharide chains. One of the carbohydrate chains is linked to an Asn residue in the sequence Asn-Ser-Cys rather than the usual Asn-X-Ser/Thr sequence. The sequence of von Willebrand factor includes several regions bearing evidence of internal gene duplication of ancestral sequences. The protein also contains the tetrapeptide sequence Arg-Gly-Asp-Ser (at residues 1744-1747), which may be a cell attachment site, as in fibronectin. The amino- and carboxyl-terminal regions of the molecule contain clusters of half-cystinyl residues. The sequence is unique except for some homology to human complement factor B.

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.

The sequence of the 841 amino acid residues in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy)which were fragmented by cleavage at methionyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder ofthe sequence. The residues involved in the covalent aind allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679, respectively.Muscle glycogen phosphorylase (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) is one of the key enzymes directly involved in the metabolism of glycogen. Upon activation through a cascade of successive enzymatic reactions, the enzyme catalyzes the conversion of glycogen and phosphate to glucose-i-phosphate. Since its isolation and crystallization (1), phosphorylase has attracted the interest of numerous investigators because it is the first enzyme in which activity was shown to be controlled by both covalent and allosteric modifications (2-4).A full understanding of the regulation and mechanism of action of the enzyme requires a detailed analysis of the structure of the subunits of the protein and of their molecular assembly. As an initial step, Saari and Fischer (5) isolated 18 fragments produced by cleavage of phosphorylase with cyanogen bromide. Titani et al. (6) showed that one of these fragments (CB14) is acetylated at its amino-terminus and that serine-14 becomes phosphorylated in the conversion of the enzyme from the b to the a form.The sequence determination described herein took advantage of a finding by Raibaud and Goldberg (7) that subtilisin cleaves the native enzyme into two reasonably homogeneous and complementary segments of molecular weight about 30,000 and 70,000. Further chemical cleavages of each of these segments at asparaginyl-glycine, aspartyl-proline, and methionyl bonds facilitated the completion of the sequence analysis. The general strategy followed for establishing the primary structure is presented herein; the experimental details will be published elsewhere. METHODS

The complete amino acid sequence of the catalytic subunit (gamma subunit) of rabbit skeletal muscle phosphorylase b kinase was determined. The gamma subunit was purified by gel filtration in acidic 8 M urea after reduction and S-carboxymethylation in 7 M guanidine hydrochloride. Cleavage of the gamma subunit at arginyl bonds gave a complete set of nonoverlapping peptides. Overlapping peptides were obtained by cleavage at methionyl, tryptophanyl, or glutamyl bonds and by selected subdigestion of two large peptides obtained by cleavage at methionyl bonds. Sequence analysis established that the protein contains 386 residues corresponding to a molecular weight (Mr) of 44673. Comparison of the gamma subunit with the catalytic subunit of bovine cAMP-dependent protein kinase and with tyrosine-specific kinases of viral origin revealed a significant degree of sequence identity among all of these proteins. These data suggest that calcium-dependent protein kinases may share a common ancestral gene and a common structural basis for catalytic function with a wide variety of other protein kinases which respond to different signals and control quite different processes.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic isolated from protein radiolabeled in vitro by ['4C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in ar-helices and 12% in a-strands.Cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) are widely distributed and play a central role in the regulation of energy metabolism and other physiological functions (1-5). In eukaryotic systems, the actions of cyclic AMP are mediated exclusively through the activation of this protein kinase. Two forms of the enzyme with slightly different regulatory properties have been isolated (6, 7): type I, found predominantly in rabbit skeletal muscle, and type II, found in bovine cardiac muscle. Both types consist of two regulatory (R) and two catalytic (C) subunits. Differences between the two are attributed to differences in the structure of the regulatory subunits; the catalytic subunits are said to be identical (8). In both types, cyclic AMP acts as a positive effector resulting in dissociation of the oligomeric protein and activation of the enzyme according to the following scheme: R2C2 (inactive) + 4 cyclic AMP -± (R-cyclic AMP2)2 + 2 C (active). As a first step toward elucidating the primary structure of the enzyme, Peters et al. (9) developed a large-scale preparation of the catalytic subunit from bovine cardiac muscle and determined its molecular weight, isoelectric point, amino acid composition, and phosphate content and the reactivity of its thiol groups. We recently described the amino acid sequence around the two sites that are phosphorylated (10). In the present communication, the sequence of the 349 amino acid residues in the catalytic subunit is presented and discussed. The experimental details of the sequence determination will be published elsewhere. METHODSThe catalytic subunit of cyclic AMP-dependent protein kinase was prepared from bovine heart as described (9). TPCK-Trypsin, pepsin, and carboxypeptidase A were purchased from Worthington. Myxobacter AL-I protease II (11) 14). Small peptides were analyzed in the sequencer in the presence of Polybrene (15). Some peptides were analyzed with a Sequemat by the method of Laursen (16) after attachment to the solid phase via carboxylte...