Peter Højrup

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K‐dependent plasma proteins reveals an extensive homology. The N‐terminal part, containing 13 γ‐carboxyglutamic acid and one β‐hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid‐102 is present. Histidine‐57 and serine‐195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.

Biochemical Evidence for Heme Linkage through Esters with Asp-93 and Glu-241 in Human Eosinophil Peroxidase THE ESTER WITH ASP-93 IS ONLY PARTIALLY FORMED IN VIVO

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857–8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.

Extractable proteins from abdominal cuticle of sexually mature locusts Locusta migratoria

Abstract The urea-extractable proteins from the abdominal cuticle of mature locusts Locusta migratoria , have been characterized by two-dimensional electrophoresis and by amino acid analysis of purified components. The existence of sex-related differences in protein composition was confirmed in mature cuticle, whereas such differences are absent in pharate cuticle. The proteins from mature cuticle are mainly acidic, while the pharate proteins are mainly basic. The deposition of the proteins characteristic for mature cuticle starts within a few days after ecdysis; the different proteins do not appear simultaneously in the extracts. It is suggested that the pharate proteins represent exocuticle, that the proteins extracted from mature cuticle are derived from endocuticle, and that the differences between the two groups of proteins will give the two layers different physical properties.

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of κ- and αs2-casein from bovine milk

Naturally occurring monomeric kappa-casein and alpha s2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin, trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (Ki) of which for inhibition of PepO was 60 μM. This peptide also inhibited (Ki=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (Ki=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aKi of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.

Localization of In Vivo Phosphorylation Sites in Multiphosphorylated Proteins

Phosphorylation of proteins is one of the most frequent forms of posttranslational modification in eukaryotic cells and is linked to the control of a multitude of cellular functions. Proteins involved in this type of regulation are typically only phosphorylated at a single or a few sites. Another type of phosphoproteins are those containing multiple phosphorylations. In these proteins the phosphorylations usually possess different functions than in proteins phosphorylated at single sites. In the case of multisite phosphorylated proteins (for review see Roach, 1991), the phosphorylations are often important as physical interactors with divalent metal ions, especially Cat2+.