Biomedical subjects

L K Rasmussen

Publications and source records attributed to L K Rasmussen.

25 records · Page 2Linked to original sources

PubMed1994

A unique interhelical insertion in plasminogen activator inhibitor-2 contains three glutamines, Gln83, Gln84, Gln86, essential for transglutaminase-mediated cross-linking.

Plasminogen activator inhibitor type 2 (PAI-2) prevents fibrinolysis by blocking plasminogen activators. It is expressed principally by trophoblast cells and macrophages. PAI-2 in trophoblast membranes has been found cross-linked to large complexes apparently catalyzed by trophoblast transglutaminase (Jensen, P. H., Lorand, L., Ebbesen, P., and Gliemann, J. (1993) Eur. J. Biochem. 214, 141-146). Recombinant human PAI-2 was labeled with [14C]putrescine catalyzed by guinea pig liver transglutaminase. The [14C]putrescine-labeled PAI-2 was digested with cyanogen bromide and trypsin, and the peptides were purified by reverse-phase high performance chromatography. Amino acid sequencing and plasma desorption mass spectrometry of the labeled peptides revealed [14C]putrescine incorporation at Gln83, Gln84, and Gln86. These residues are present in a PAI-2-specific region of 33 amino acids that is inserted between helices C and D and which probably represents a unique solvent-exposed domain. A PAI-2 mutant lacking this insertion was determined not to be a substrate for transglutaminase by [14C]putrescine incorporation and could not form transglutaminase-catalyzed polymers. Thus, the unique PAI-2 insertion represents a functional domain that, by virtue of its transglutaminase acceptor sites, allows participation in binding reactions without affecting the inhibitory function of PAI-2.

Amino Acid Sequence↗

PubMed1994

The plasminogen activation system in bovine milk: differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor.

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of kappa-casein and dimeric alpha s2-casein were identified as t-PA binding components. The kappa-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229-236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Animals↗

PubMed1994

Identification of glutamine and lysine residues in Alzheimer amyloid beta A4 peptide responsible for transglutaminase-catalysed homopolymerization and cross-linking to alpha 2M receptor.

The beta-amyloid peptide (beta A4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimer's disease. Here we report that the full-length (1-40) synthetic beta A4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase-mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of beta A4 with alpha 2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site-specific probes followed by enzymatic digestion and sequencing of tracer-containing fractions demonstrated that both Lys16, Lys28 and Gln15 in beta A4 were susceptible to cross-linking by transglutaminase.

Alzheimer Disease↗

PubMed1994

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of kappa- and alpha s2-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.

Amino Acid Sequence↗

PubMed1992

The multimeric structure and disulfide-bonding pattern of bovine kappa-casein.

Bovine kappa-casein was analyzed by SDS/PAGE, MS and amino acid sequence analysis in order to determine its multimeric composition and disulfide-bonding pattern. SDS/PAGE revealed that kappa-casein in the native state can range in size from a monomer to a multimeric structure larger than a decamer. Three types of interchain disulfide linkage, Cys11-Cys11, Cys11-Cys88 and Cys88-Cys88, were all assigned in multimers purified from [14C]carboxymethylated and untreated bulk milk, as well as a milk sample from a kappa-casein-variant-B homozygote Co20. These results indicate that multimerization occurs in a random or at present unpredictable disulfide-bonding pattern regardless of the size of the multimer or the genotype.

Amino Acid Sequence↗

PubMed1992

Localization of two interchain disulfide bridges in dimers of bovine alpha s2-casein. Parallel and antiparallel alignments of the polypeptide chains.

Carboxymethylation of bovine skimmed milk with 14C-labelled iodoacetic acid followed by purification of the alpha s2-casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine-containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide-linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide-linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.

Amino Acid Sequence↗

PubMed1991

Purification of disulphide-linked alpha s2- and kappa-casein from bovine milk.

Naturally occurring disulphide-linked alpha s2- and kappa-casein in bovine milk were purified by gel chromatography on a column of Sepharose CL-6B. Four fractions (A-D) were obtained by elution with ammonium acetate-urea buffer. Fractions A and B, identified by SDS gel electrophoresis and amino acid sequence analysis, corresponded to disulphide-linked kappa-casein and alpha s2-casein respectively. Fraction C consisted of a mixture of alpha s1-, alpha s2-, and beta-casein. Separation of fraction C into its components was achieved by reversed-phase HPLC. The stability of the disulphide bridges in alpha s2- and kappa-casein was shown to differ with respect to reducing agents (dithioerythritol and 2-mercaptoethanol).

Amino Acid Sequence↗