scholarly journals Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

1990 ◽  
Vol 171 (2) ◽  
pp. 565-570 ◽  
Author(s):  
K Ritter ◽  
H Brestrich ◽  
B Nellen ◽  
H Kratzin ◽  
H Eiffert ◽  
...  
Keyword(s):  
Infectious Mononucleosis ◽  
Triosephosphate Isomerase ◽  
Clinical Symptoms ◽  
Glycolytic Enzyme ◽  
Amino Acid Sequences ◽  
Cellular Proteins ◽  
51Cr Release ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

2000 ◽  
Vol 83 (03) ◽  
pp. 438-444 ◽  
Author(s):  
Claudio Sampaio ◽  
Reinhard Mentele ◽  
Antonio Camargo ◽  
Edwin Fink ◽  
Solange Serrano
Keyword(s):  
Amino Acid Sequences ◽  
Amidolytic Activity ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Bothrops Jararaca ◽  
Sds Page ◽  
Competitive Inhibitors ◽  
Molecular Masses ◽  
Terminal Amino ◽  
Serine Endopeptidases

SummaryThree chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

scholarly journals β-Galactosidase II in Ripening Tomatoes

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 817A-817
Author(s):  
Russell Pressey ◽  
C.M. Sean Carrington
Keyword(s):  
Amino Acid ◽  
Cell Walls ◽  
Amino Acid Sequences ◽  
Cell Wall Polysaccharides ◽  
Terminal Amino Acid ◽  
Molecular Weights ◽  
Sds Page ◽  
Original Procedure ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Tomatoes contain several isozymes of β-galactosidase, but only one, β-galactosidase II, can hydrolyze the β-1,4-galactans in tomato cell walls. β-galactosidase II has now been highly purified by modification of the original procedure. The molecular weight of this isozyme is ≈62 kDa according to gel infiltration, but SDS-PAGE of the purified enzyme separated three components with molecular weights of 29, 42, and 82 kDa. The 82-kDa peptide may be the intact enzyme and the smallest peptides are subunits as proposed for other β-galactosidases. The N-terminal amino acid sequence of β-galactosidase II showed high homology with amino acid sequences reported for other plant β-galactosidases. A new assay for β-galactosidase II in tomato extracts has been developed using FPLC. This isozyme was not detected in mature-green tomatoes but appeared at about the breaker stage and increased during ripening. The increase in b-galactosidase II was accompanied by a decrease in galactose content of cell wall polysaccharides, suggesting that this enzyme may be involved in the loss of galactose during tomato ripening.

scholarly journals 1,2-α-d-mannosidase from Penicillium citrinum: molecular and enzymic properties of two isoenzymes

1993 ◽  
Vol 290 (2) ◽  
pp. 349-354 ◽  
Author(s):  
T Yoshida ◽  
T Inoue ◽  
E Ichishima
Keyword(s):  
Amino Acid ◽  
Gel Permeation Chromatography ◽  
Amino Acid Sequences ◽  
Penicillium Citrinum ◽  
Tryptic Digestion ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino ◽  
Hydrolysis Of ◽  
Similar Kinetic

Two isoforms of acidic 1,2-alpha-D-mannosidases have been isolated from culture filtrate of Penicillium citrinum. The pI values of the two forms, designated 1,2-alpha-mannosidase Ia and Ib, were 4.6 and 4.7 respectively. Isoenzymes Ia and Ib exhibited the same molecular mass which was determined to be 53 kDa by SDS/PAGE and 54 kDa by gel-permeation chromatography. Enzymes Ia and Ib hydrolysed yeast mannan and 1,2-alpha-linked mannooligosaccharides, but did not hydrolyse p-nitrophenyl alpha-D-mannoside. The optimal pH for the hydrolysis of Man(alpha 1-->2)Man was 5.0 for both isoenzymes. Similar kinetic parameters were determined for the two forms. Activation energy was a little lower for Ia than Ib. There was little difference between the enzymes with regard to their performance at acidic or alkaline pH. The N-terminal amino acid sequences of the two enzymes were identical. Analysis of C-terminal peptides, which were prepared by tryptic digestion and anhydrotrypsin-agarose chromatography, showed that Ia and Ib had the same amino acid sequences in the C-terminal region. Tryptic digestion revealed a slight difference between the isoenzymes in the pattern of cleaved peptides on SDS/PAGE.

scholarly journals Purification and characterization of two forms of β-d-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s)

1995 ◽  
Vol 305 (1) ◽  
pp. 41-50 ◽  
Author(s):  
D R P Tulsiani ◽  
M D Skudlarek ◽  
Y Araki ◽  
M C Orgebin-Crist
Keyword(s):  
Plasma Membrane ◽  
Amino Acid Sequences ◽  
Molecular Forms ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Before And After ◽  
Sperm Plasma Membrane ◽  
Terminal Amino ◽  
Ph Dependent

Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Factor D̄ of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site

1980 ◽  
Vol 187 (3) ◽  
pp. 863-874 ◽  
Author(s):  
D M Johnson ◽  
J Gagnon ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Serine Residue ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat ‘group-specific protease’ [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.

Comparative analyses of Serratia spp. outer membrane porin proteins

1993 ◽  
Vol 39 (4) ◽  
pp. 442-447 ◽  
Author(s):  
Joanne Hutsul ◽  
Elizabeth Worobec ◽  
Tom R. Parr Jr. ◽  
Gerald W. Becker
Keyword(s):  
Amino Acid ◽  
Serratia Marcescens ◽  
Single Channel ◽  
Enteric Bacteria ◽  
Amino Acid Sequences ◽  
Chromosomal Dna ◽  
Terminal Amino Acid ◽  
Molecular Weight Range ◽  
Terminal Amino ◽  
Porin Proteins

Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunoblot and Southern DNA hybridization experiments and probed with antibody and DNA probes specific for the 41-kDa Serratia marcescens porin, to determine the extent of homology between Gram-negative porins. Immunoblot analyses performed using porin-specific rabbit sera and cell envelope preparations from these strains revealed that all strains produced at least one cross-reactive protein in the 41-kDa molecular weight range. Chromosomal DNA from each of the same strains was used in Southern analyses, probed with a 20-base-length oligonucleotide probe deduced from the N-terminal amino acid sequence of the 41-kDa Serratia marcescens porin. The probe hybridized to DNA from all of the Serratia species and six of the nine other enteric bacteria. Putative porin proteins from all the Serratia species were subjected to N-terminal amino acid sequencing and porin functional analysis using the black lipid bilayer method. All amino acid sequences were identical, with one exception in which an asparagine was substituted for an aspartic acid in Serratia rubidaea. All porins had very similar porin function (single channel conductance ranging between 1.72 and 2.00 nS). The results from this study revealed that a strong conservation exists among the Serratia porins and those produced by other enteric bacteria.Key words: porins, Serratia marcescens, homology studies.

Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid

Biochemistry ◽  
1978 ◽  
Vol 17 (3) ◽  
pp. 442-445 ◽  
Author(s):  
Mark A. Hermodson ◽  
Kirk C. S. Chen ◽  
Thomas M. Buchanan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Amino Acid ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Unusual Amino Acid
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