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.

No abstract

The method of sequenator analysis described by Edman and Begg (Edman, P., and Begg, G. (1967), Eur. J . Biochem. 1, 80) has been modified and applied to proteins and protein fragments. Significant modifications include the replacement of Quadrol by a volatile buffer (dimethylbenzylamine), the introduction of thiols to stabilize the reaction products, and the identification of the reaction products as silylated phenylthiohydantoins by automated gas-liquid chromatography. With these and other modifications, 30-50 T he sequential degradation of peptides by the method of Edman (1956) is an important procedure for the determination of amino acid sequences of proteins. The method combines the specificity of end-group analysis with the advantages of a cyclic stepwise process and normally yields 7-15 unambiguous degradations. In 1967, Edman and Begg automated the process by designing an instrument called the "sequenator" and demonstrated its successful application to the identification of 60 amino-terminal residues of apomyoglobin. Since then, other sequenators have been constructed, built on the principles of Edman and Begg. According to published accounts, these instruments are capable of Smithies et a/., Titani et al., 1972a).The capability of the sequenator to determine long amino acid sequences has altered the general strategy of sequence analysis. Instead of fragmenting the protein into a large number of short peptides whose sequences can be determined by manual Edman degradations and by digestion with carboxypeptidases, the protein is cleaved into a small number of large fragments, usually by chemical procedures (e.g., cyanogen bromide, hydroxylamine), and the separated fragments are directly subjected to automated sequence analysis. Only those segments which cannot be reached by the sequenator are subsequently analyzed by the classical procedures.Sequenator analysis has also been effective for screening proteins for homology, simply by applying the sequential analysis to the amino-terminal region of the protein or to other regions adjacent to existing or newly created a-amino groups. Such initiation points for consecutive degradations can be established by chemical reactions or by limited enzymatic proteolysis.Because of its sensitivity and the small amount of protein required for but a few turns, sequenator analysis is a rapid and amino acid residues can be identified and recovered with a repetitive yield of approximately 96 %. This modified method has been tested on thermolysin and its cyanogen bromide fragments and found to be reliable in determining amino acid sequences. It has also been applied to porcine trypsin and found to be of use in determining purity, allotypic variants, and internal peptide-bond cleavage. In addition, the chemical nature of protein subunits can be identified by this method.accurate test for protein purity and, inter alia, for determining the number of polypeptide chains in a pure oligomeric protein. The method also has proved useful in following the changes in covalent structure att...

Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factoractivated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp9(k) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation.The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.Microtubule-associated protein 2 kinases (MAP kinases) belong to a group of protein serine/threonine kinases that are activated in response to extracellular stimuli in a variety of cell types (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) cDNA encoding a homologous kinase, ERK2, which is 90% identical to ERK1 (19). ERK1 and ERK2 were both shown to be activated in vivo by growth factors, concomitant with their phosphorylation on tyrosine and threonine residues (16,17,19). In the present report, experiments with purified, low activity MAP kinase preparations (ERK1 and ERK2) reveal that phosphate incorporation into tyrosine and threonine residues of each kinase can occur as a result of a selfcatalyzed reaction of the enzyme itself. The reaction is slow compared to that which occurs in the presence ofMAP kinase activator but is accompanied by activation of the enzyme. This surprising result suggests that autophosphorylation may be involved in the MAP kinases activation process and emphasizes the possibility that MAP kinase activator need not necessarily possess kinase activity itself. MATERIALS AND METHODSProteins. Recombinant ERK2 was purified from Escherichia coliusing fractionation steps ofAffi-Gel blue followed by DEAEcellulose as described by Boulton et aL (19). The ERK2 obtained was judged by silver staining to be >90% pure. ERK1 was purified from insulin-stimulated Rat 1 HIRcB cells up to the DEAE-cellulose step as described by Boulton et al. (17) and contained mostly this enzyme but also some (4106) ERK2. MAP kinase activator from epidermal growth factor-stimulated A431 cells was partially purified by using the Mono Q and Mono S steps as described by Ahn et aL (14).Kinase Assays. MAP kinase activity was determined by phosphate incorporation into myelin basic protein (MBP) as described (5, tTo whom reprint requests should be a...

Photolyzed rhodopsin is phosphorylated at multiple serine and threonine residues during the quenching of phototransduction. Sites of phosphorylation by rhodopsin kinase have been localized to the C-terminal region of rhodopsin, but no information was available on the kinetics and identity of phosphorylated residues. To determine the kinetics of phosphorylation at specific residues, the phosphorylated C-terminal peptide of rhodopsin (330DDEASTTVSKTETSQVAPA) obtained by proteolysis of rhodopsin with endoproteinase Asp-N was subjected to further subdigestion followed by electrospray mass spectrometry. Analysis of monophosphorylated peptide revealed that the major initial phosphorylation site is 338Ser. The analysis of di- and triphosphorylated peptides indicated that 343Ser or 336Thr residues are subsequent phosphorylation sites. These three residues, located in the C-terminal region of rhodopsin, are likely to be key phosphorylation sites of rhodopsin during the quenching of phototransduction. Identification of the kinetics of phosphorylation will facilitate understanding the functional significance of rhodopsin phosphorylation at multiple sites and the mechanism of rhodopsin kinase action.