scholarly journals Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences

1995 ◽  
Vol 307 (2) ◽  
pp. 341-346 ◽  
Author(s):  
S S Little ◽  
D A Johnson
Keyword(s):  
Mast Cell ◽  
Molecular Mass ◽  
Peptide Mapping ◽  
Proteinase Inhibitors ◽  
High Molecular Mass ◽  
Mass Separation ◽  
Human Mast Cell ◽  
Mast Cell Tryptase ◽  
Sds Page ◽  
Mass Form

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.

1,2-Benzisothiazol-3-one 1,1-Dioxide Inhibitors of Human Mast Cell Tryptase

1998 ◽  
Vol 41 (24) ◽  
pp. 4854-4860 ◽  
Author(s):  
Keith D. Combrink ◽  
H. Belgin Gülgeze ◽  
Nicholas A. Meanwell ◽  
Bradley C. Pearce ◽  
Pi Zulan ◽  
...  
Keyword(s):  
Mast Cell ◽  
Human Mast Cell ◽  
Mast Cell Tryptase

Human mast cell tryptase attenuates the vasodilator activity of calcitonin generelated peptide

1992 ◽  
Vol 43 (6) ◽  
pp. 1243-1248 ◽  
Author(s):  
Andrew F. Walls ◽  
Susan D. Brain ◽  
Anita Desai ◽  
Peter J. Jose ◽  
Elaine Hawkings ◽  
...  
Keyword(s):  
Mast Cell ◽  
Human Mast Cell ◽  
Mast Cell Tryptase ◽  
Vasodilator Activity

Human mast cell tryptase: a biochemical marker for mast cell degranulation

1989 ◽  
Vol 17 (4) ◽  
pp. 728-729 ◽  
Author(s):  
ANDREW F. WALLS ◽  
AMANDA R. BENNETT ◽  
HARRY M. McBRIDE ◽  
MARTIN J. GLENNIE ◽  
STEPHEN T HOLGATE ◽  
...  
Keyword(s):  
Mast Cell ◽  
Biochemical Marker ◽  
Mast Cell Degranulation ◽  
Human Mast Cell ◽  
Mast Cell Tryptase

Dibasic inhibitors of human mast cell tryptase. Part 1: Synthesis and optimization of a novel class of inhibitors

2000 ◽  
Vol 10 (20) ◽  
pp. 2357-2360 ◽  
Author(s):  
Kenneth D Rice ◽  
Anthony R Gangloff ◽  
Elaine Y.-L Kuo ◽  
Jeffrey M Dener ◽  
Vivian R Wang ◽  
...  
Keyword(s):  
Mast Cell ◽  
Human Mast Cell ◽  
Mast Cell Tryptase

scholarly journals Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the ‘26 S’ multienzyme complex

1991 ◽  
Vol 280 (1) ◽  
pp. 225-232 ◽  
Author(s):  
A Seelig ◽  
P M Kloetzel ◽  
L Kuehn ◽  
B Dahlmann
Keyword(s):  
Molecular Mass ◽  
Molecular Interaction ◽  
Gradient Centrifugation ◽  
Mass Range ◽  
High Molecular Mass ◽  
Multienzyme Complex ◽  
Multicatalytic Proteinase ◽  
Sds Page ◽  
Specific Antisera ◽  
Rabbit Reticulocytes

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a ‘26 S’ complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass ‘26 S’ multienzyme complex. In all instances proteasomes are identified in their ‘free’ 650 kDa ‘20 S’ form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described ‘26 S’ proteinase. This ‘26 S’ proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the ‘26 S’ proteinase complex.

The allosteric effect of salt on human mast cell tryptase

1988 ◽  
Vol 956 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Ilkka T. Harvima ◽  
Rauno J. Harvima ◽  
Terho O. Eloranta ◽  
Jorma E. Fräki
Keyword(s):  
Mast Cell ◽  
Human Mast Cell ◽  
Allosteric Effect ◽  
Mast Cell Tryptase

scholarly journals Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization

1994 ◽  
Vol 304 (3) ◽  
pp. 693-698 ◽  
Author(s):  
L W J Klomp ◽  
L van Rens ◽  
G J Strous
Keyword(s):  
Molecular Mass ◽  
Mechanical Damage ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
Apparent Molecular Mass ◽  
Gastric Mucin ◽  
Gastric Mucus ◽  
Apical Side ◽  
Sds Page ◽  
Glycosylation Inhibitor

Gastric mucin plays an important role in the protection of the stomach wall from chemical, microbiological and mechanical damage. We have previously isolated human gastric mucus glycoproteins and raised a polyclonal antiserum against these macromolecules. This antiserum specifically reacted with gastric mucins in immunoblotting experiments and stained mucous granules at the apical side of gastric surface epithelial cells. A similar staining pattern was obtained after incubation with an antiserum against rat gastric mucin. Next we used the antiserum in pulse-chase experiments of human stomach tissue explants. After short labelling periods with [35S]methionine and [35S]cysteine, the antiserum reacted with a polypeptide with an apparent molecular mass of approx. 500 kDa as determined by SDS/PAGE, which was converted after 90 min into a heterogeneous high-molecular-mass glycoprotein. This high-molecular-mass form, but not the 500 kDa polypeptide, was detectable in the culture medium after 2 h. This strongly suggests that the 500 kDa polypeptide is the precursor of the purified gastric mucin. Analysis of pulse-chase experiments by non-reducing SDS/PAGE revealed that the precursors form disulphide-linked oligomers early in biosynthesis, before the addition of O-linked sugars. After preincubation with the N-glycosylation inhibitor, tunicamycin, the apparent molecular mass of the precursor decreased marginally but consistently, indicating that N-linked glycan chains are present on the mucin precursor.

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