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.

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.

Triticum aestivum L. endoxylanase inhibitor (TAXI) consists of two inhibitors, TAXI I and TAXI II, with different specificities

2001 ◽  
Vol 353 (2) ◽  
pp. 239-244 ◽  
Author(s):  
Kurt GEBRUERS ◽  
Winok DEBYSER ◽  
Hans GOESAERT ◽  
Paul PROOST ◽  
Jozef VAN DAMME ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Evolutionary Relationship ◽  
Triticum Aestivum L ◽  
Amino Acid Sequences ◽  
Molecular Structures ◽  
Molecular Forms ◽  
Inhibition Activity ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
High Degree

The Triticum aestivum L. endoxylanase inhibitor (TAXI) discovered by Debyser and Delcour [(1997) Eur. Pat. filed April 1997, published as WO 98/49278] and Debyser, Derdelinckx and Delcour [(1997) J. Am. Soc. Brew. Chem. 55, 153Ő156] seems to be a mixture of two different endoxylanase inhibitors, called TAXI I and TAXI II. By using Aspergillus niger as well as Bacillus subtilis endoxylanases for assaying inhibition activity, both inhibitors could be purified to homogeneity from wheat (Triticum aestivum L., var. Soissons). TAXI I and TAXI II have similar molecular structures. They both have a molecular mass of approx. 40.0kDa, are not glycosylated and occur in two molecular forms, i.e. a non-proteolytically processed one and a proteolytically processed one. However, the pI of TAXI II (at least 9.3) is higher than that of TAXI I (8.8). TAXI I and TAXI II clearly show different inhibition activities towards different endoxylanases. The N-terminal amino acid sequences of both inhibitors show a high degree of identity, which might indicate that there is an evolutionary relationship between them.

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 Identification of one- and two-chain forms of trypsinogen 1 produced by a human gastric adenocarcinoma cell line

1994 ◽  
Vol 303 (1) ◽  
pp. 187-190 ◽  
Author(s):  
N Koshikawa ◽  
H Yasumitsu ◽  
Y Nagashima ◽  
M Umeda ◽  
K Miyazaki
Keyword(s):  
Gastric Adenocarcinoma ◽  
Amino Acid Sequences ◽  
Molecular Forms ◽  
Adenocarcinoma Cell ◽  
Sds Page ◽  
Human Gastric Adenocarcinoma ◽  
Proteolytic Activities ◽  
Terminal Amino ◽  
The One ◽  
Chain Form

It has previously been reported that two kinds of human gastric adenocarcinoma cell lines (STKM-1 and MKN28) secrete a trypsin-like enzyme. In this study, four molecular forms of the enzyme (26, 25, 24 and 23 kDa on non-reducing SDS/PAGE) were purified from the serum-free conditioned medium of STKM-1 cells. Analysis of N-terminal amino acid sequences showed that the 26 kDa protein was a two-chain form of trypsinogen 1 which had been produced by proteolytic cleavage of the Arg107-Val108 bond of trypsinogen 1, and the 24 kDa protein was the one-chain form of trypsinogen 1. The 25 and 23 kDa proteins were the activated forms of the two-chain and one-chain trypsinogen 1 respectively. Isoelectric focusing gave pI values of 6.3 and 6.6 for the 26 kDa two-chain form and the 24 kDa one-chain form of trypsinogen 1 respectively. Comparison of the proteolytic activities indicated that the one-chain trypsin 1 had amidolytic activity about four times higher than that of the two-chain enzyme.

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.

A major Litomosoides carinii microfilarial sheath glycoprotein (gp22): amino terminal sequence and immunological studies with corresponding synthetic peptides

Parasitology ◽  
1991 ◽  
Vol 103 (3) ◽  
pp. 387-394 ◽  
Author(s):  
G. Bardehle ◽  
F. J. Conraths ◽  
F. Fahrenholz ◽  
M. Hintz ◽  
D. Linder ◽  
...  
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
General Pattern ◽  
Amino Sugar ◽  
Amino Acid Sequences ◽  
Terminal Amino Acid ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Amino Terminal Sequence

The major glycoprotein of the sheath of Litomosoides carinii microfilariae (gp22) was analysed for its amino acid and amino sugar composition. It is rich in proline, glutamine/glutamic acid and glycine and contains (N-acetyl)galactosamine. The N-terminal amino acid sequence was determined up to position 37. It consists of a group of 6 repeats of the pentapeptide sequence methionine-glycine-proline-glutamine-proline with two minor modifications in repeats 3–6, while the first two repeats follow the general pattern more loosely. Identical N-terminal amino acid sequences were found in at least two other sheath polypeptides (33 kDa, 39 kDa). Antisera prepared against 3 overlapping synthetic peptides corresponding to the amino terminus of gp22 recognized different epitopes. They all reacted with identical patterns of sheath polypeptides. The antisera failed to recognize antigens of 4th-stage larvae of L. carinii. In contrast, cross-reacting epitopes were detected in other parasite stages. Antisera reacted with material surrounding embryos and microfilariae in the uterus of females, and caused patchy fluorescence on the sheath of blood-derived and in vitro-released microfilariae.

THE USE OF REGION SPECIFIC RADIOIMMUNOASSAYS FOR CHARACTERIZATION OF CIRCULATING CALCITONIN IN PATIENTS WITH MEDULLARY CARCINOMA OF THE THYROID GLAND

1979 ◽  
Vol 91 (3) ◽  
pp. 449-461 ◽  
Author(s):  
Lisbeth Myhre ◽  
Kaare M. Gautvik
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Amino Terminal ◽  
Terminal Fragment ◽  
Terminal Amino ◽  
Carboxy Terminal ◽  
Amino Terminal Fragment

ABSTRACT Two antisera with known region specificities have been used to characterize calcitonin immunoreactivity (iCT) in serum of patients with medullary thyroid carcinoma (MCT). Antiserum I which was raised against the synthetic hormone (1–32 amino acid residues), contained heterogeneous populations of immunoglobulins directed predominantly against carboxyterminal sequences of the hormone, but the antiserum reacted also with the amino-terminal fragment (1–10 amino acid residues). Antiserum II, which was raised against the carboxy-terminal hormone fragment (11–32 amino acid residues) reached equally well with the intact hormone and the C-terminal fragment, but showed negligible binding of the amino terminal fragment. Antiserum I measured therefore both amino-terminal and carboxy-terminal sequences of calcitonin while antiserum II measured only carboxy-terminal amino acid sequences. In 40 patients with MCT, antiserum I measured usually the highest concentration of serum iCT suggesting the presence of non-uniform hormone immunoreactivity. The different molecular forms of circulating iCT in 7 MCT patients were explored by using antiserum I after gel filtration on Sephadex G-100. The patients who were selected on basis of iCT measurement in serum using antiserum I and II, could be divided into 3 groups which showed characteristic iCT profiles. Group 1, in which antiserum II measured a higher concentration of serum iCT, contained predominantly (60–70 %) small fragments of calcitonin immunoreactivity. On the other hand, in the sera of group 3 in which antisera I measured an equal or the highest concentrations, the dominant form of the hormone consisted of molecular sequences equal to or larger than the intact hormone (90 %). In group 2, the two antisera measured an equal amount of serum iCT and molecular forms consisting mostly of larger hormone fragments dominated (50 %). All the patients were normocalcaemic in spite of frequently grossly elevated serum iCT, and 33 out of 36 patients had normal serum immunoreactive parathyroid hormone. In conclusion: 1. Serum iCT is heterogeneous and represents peptides of quite different molecular size with no or low biological activity. 2. Most of the serum calcitonin immunoreactivity consists of peptides with carboxy-terminal amino acid sequences. 3. Most, if not all, of the amino-terminal calcitonin immunoreactivity is due to monomeric and polymeric hormonal forms.

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.

Role of the amino-terminal amino acid sequences determining the in vitro refolding process of prochymosin polypeptide

1993 ◽  
Vol 28 (1) ◽  
pp. 85-97 ◽  
Author(s):  
Minoru Yonezawa ◽  
Junko Suzuki ◽  
Makoto Nishiyama ◽  
Sueharu Horinouchi ◽  
Teruhiko Beppu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Amino Acid ◽  
Amino Terminal ◽  
Terminal Amino ◽  
In Vitro Refolding

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.

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