Lowell H. Ericsson

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross‐linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecules HMW chains. This peptide gave two amino acid sequences (in ratio) consistent with it being a three‐chained structure. The major sequence matched exactly that of the C‐telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross‐links also gave two amino‐terminal sequences, one from its own amino terminus, the other matching exactly the N‐telopeptide cross‐linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross‐linked in cartilage to molecules of type II collagen, probably at fibril surfaces.

The major proteins of human and monkey amyloid substance: Common properties including unusual N-terminal amino acid sequences

Amyloid substance is a complex proteinaceous material found in the tissues of patients with the disease amyloidosis. Recently we have presented evidence that there are two chemically distinct kinds of amyloid substance, one associated with the classical inflammation-related amyloidosis and another, frequently designated atypical or paramyloid, occurring with tumors such as multiple myeloma or without evident pre-existing disease [l] . The classical, or inflammation-associated, substance is distinguished by: a) instability to alkali of its characteristic Congo red binding capacity; b) its amino acid composition and c) the presence of a major protein constituent, amyloid protein A. The family of proteins comprising amyloid protein A has a molecular weight range of 6000-8000 and a characteristic amino acid composition [2] ; in addition, human amyloid protein A has the capacity to bind Congo red and exhibits the characteristic hyperchromism and spectral changes previously described for amyloid substance [ 1] . In this communication we compare the chromatographic and electrdphoretic properties, the amino acid composition and the 24 amino acid N-terminal sequence of the humanand monkeyderived protein A of amyloid substance.

Identification of fatty acids by electrospray mass spectrometry and tandem mass spectrometry

Low-energy negative-ion electrospray mass spectrometry (ESI-MS) and ESI-MS/MS were used to characterize saturated and unsaturated fatty acids. The carbon number and degree of unsaturation of fatty acids were determined using ESI-MS, and MS/MS was used to localize some double bond positions of mono-and polyunsaturated fatty acids. For compounds with up to two unsaturated bonds, fragmentation was dominated by loss of H2O from the carboxyl moiety and very low-intensity peaks generated from bonds cleaved at carbons alpha and/or beta to sites of unsaturation. Fragmentation of monounsaturated fatty acids was minimal using this soft method of mass spectrometric analysis, but increased with progressively greater degrees of fatty acid unsaturation. There was extensive hydride migration during ESI-MS/MS of compounds with three or more double bonds. Although this behavior complicated localization of double and triple bonds, the spectra were reproducible. Many peaks could not be definitively assigned to specific product ions, but the spectra of standards and complementary natural products were similar and isobaric compounds could be differentiated. The utility of this technique to examine biological samples was shown by analysis of the fatty acid composition of cod liver oil. Detection limits for negative-ion ESI-MS/MS were at or below 1 pg.

Primary structure of duck amyloid protein A The form deposited in tissues may be identical to its serum precursor

The amino acid sequence has been determined for the major protein that accumulates in amyloid fibrils in tissues of the Pekin duck. With the exception of 16 residues at the amino terminus, this 106‐residue protein is homologous with human serum amyloid protein A (104‐residue apoSAA), which is the putative precursor of the 76‐residue protein that accumulates in human patients with amyloidosis. Duck serum is shown to contain a protein that is immunologically related and approximately equal in size (12 kDa) to the deposited form in ducks. These results indicate that proteolytic processing of the precursor is not a necessary step in the deposition of amyloid fibrils, at least in the duck.

Primary structure of yeast cytochrome c peroxidase: II. The complete amino acid sequence☆☆☆

Abstract The complete amino acid sequence of 293 residues in the single polypeptide chain of yeast cytochrome c peroxidase has been determined. Sequence analyses were performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion of citraconylated protein. These fragments were aligned with sequential and compositional data as well as those obtained with intact protein, tryptic, and chymotryptic peptides (Takio and Yonetani, 1980, Arch. Biochem. Biophys. 203 , 605–614). Residues critical for catalysis by the enzyme were identified as arginine 48, tryptophan 51, histidine 52, and histidine 174 by fitting the sequence to the electron density map derived by others. Sequence comparison with other proteins show limited homology with horseradish peroxidase and myoglobin.

Phosphorylation of glycogen synthase by phosphorylase kinase. Stoichiometry, specificity and site of phosphorylation

The enzyme glycogen synthase contains multiple phosphorylation sites per tetrameric subunit which can be phosphorylated by CAMP-dependent and CAMP-independent protein kinases (reviewed [ 11). On the basis of analysis of tryptic and CNBr [“PIpeptides we have defined two phosphorylation domains per 90 000 dalton subunit [2]. The trypsinsensitive domain of Mr 17 000 near the subunit Cterminus contains two or more phosphorylation sites [3,4] which are preferentially phosphorylated by the CAMP-dependent protein kinase. The trypsin-insensitive domain of 10 000 daltons is preferentially phosphorylated by a CAMP-independent synthase kinase [2]. Recently, a CDRdependent synthase kinase has been reported [S ,6] which now appears to be identical with phosphorylase kinase which can also catalyze phosphorylation and inactivation of glycogen synthase [7-91. In this paper, antiserum to phosphorylase kinase was used to confirm the conclusion that phosphorylase kinase itself catalyzes phosphorylation of glycogen synthase. It is also shown that the presence of phosphorylase inhibits the inactivation of glycogen synthase by phosphorylase kinase and that the phosphorylation site in the trypsin-insensitive

Amino acid sequence of Streptomyces griseus protease B, a major component of pronase

Summary Streptomyces griseus Protease B is a close homologue of Protease A, another serine protease isolated from Pronase. Homology based on identity of residues in the two enzymes is 61%. Extensive identical sequences are found in the vicinities of histidine-57, aspartic acid-102, serine-195, the two disulfide bridges, the NH2-terminal and COOH-terminal ends as well as in the region of the presumed substrate binding sites. However, certain regions of the Protease B sequence are markedly different and include a heptapeptide insertion between residues 88 and 89 and a tetrapeptide deletion between residues 129 and 136 when compared with Protease A. These differences are probably responsible for the remarkable stability of Protease B in concentrated solutions of urea and guanidine hydrochloride.

Phosphorylation of human gp130 at Ser-782 adjacent to the Di-leucine internalization motif. Effects on expression and signaling.

The receptor for leukemia inhibitory factor (LIF) consists of two polypeptides, the LIF receptor and gp130. Agonist stimulation has been shown previously to cause phosphorylation of gp130 on serine, threonine, and tyrosine residues. We found that gp130 fusion proteins were phosphorylated exclusively on Ser-782 by LIF- and growth factor-stimulated 3T3-L1 cell extracts. Ser-780 was required for phosphorylation of Ser-782 but was not itself phosphorylated. Ser-782 is located immediately N-terminal to the di-leucine motif of gp130, which regulates internalization of the receptor. Transient expression of chimeric granulocyte colony-stimulating factor receptor (G-CSFR)-gp130(S782A) receptors resulted in increased cell surface expression in COS-7 cells and increased ability to induce vasoactive intestinal peptide gene expression in IMR-32 neuroblastoma cells when compared with expression of chimeric receptors containing wild-type gp130 cytoplasmic domains. These results identify Ser-782 as the major phosphorylated serine residue in human gp130 and indicate that this site regulates cell surface expression of the receptor polypeptide.

Bovine factor IX (christmas factor). Further evidence of homology with factor X (Stuart factor) and prothrombin

Factor IX is a single-chain glycoprotein involved in the intrinsic pathway of blood coagulation. The molecular characteristics of factor IX and the mechanism of its activation by factor XI, (activated factor XI) have recently been described [ 1,2] . In the presence of factor VIII, calcium, and phospholipid, factor IX, catalyzes the conversion of factor X to X,. Factor X, in the presence of factor V, calcium, and phospholipid in turn converts prothrombin to thrombin. Factor X, and thrombin are both inactivated by diisopropylphosphorofluoridate (DFP), but the same has not been established for factor IX,. Factor X, and thrombin are homologous with other mammalian serine proteases [3]. The amino terminal regions of factor IX, prothrombin, and the light chain of factor X are also homologous, but factor IX is immunologically distinct [4]. Nevertheless, evidence is presented here showing that considerable homology exists between bovine factors IX and X and prothrombin, suggesting that factor IX, has evolved from the same precursor and therefore is probably also a serine protease.

The amino-terminal sequence of an invertebrate trypsin (crayfish Astacus leptodactylus): Homology with other serine proteases

The serine proteases represent one of the best characterized families of proteins which have evolved from a common ancestor [ 11. They include such functionally diverse and distinct enzymes as the digestive proteases of the pancreas, trypsin, chymotrypsin and elastase, and the hepatic proteases of the blood coagulation system, thrombin, factor X, and factor IX, [2]. Plasmin [ 1) and kallikrein [3] also belong to this family. Among these enzymes, the trypsins, defined by common features of their active site, seem to have been preserved during the whole span of evolution ranging from bacteria to mammals. The complete covalent structures of three vertebrate trypsins, i.e., bovine [4], porcine [S] and dogfish [6] have been reported and structural details of several other vertebrate trypsins are known in part [4]. In contrast, little information is available on trypsins of invertebrate animals despite the fact that nine of ten phyla of the animal kingdom are represented by invertebrates. A proteolytic enzyme which exhibits tryptic specificity toward peptides and synthetic substrates [7,8] was found in the invertebrate crayfish Astacus leptoductylus and was later identified as a serine protease [9]. In contrast to bovine trypsin, the crayfish trypsin is a rather acidic protein which is irreversibly inactivated at low pH [8]. The molecular weight, calculated from the amino acid composition is 24 000 [lo] and in contrast to the vertebrate trypsins crayfish trypsin contains 6 rather than 12 half-cystine residues. The en-