scholarly journals Human leucocyte glycosylasparaginase is an α/β-heterodimer of 19 kDa α-subunit and 17 and 18 kDa β-subunit

1994 ◽  
Vol 300 (2) ◽  
pp. 541-544 ◽  
Author(s):  
O K Tollersrud ◽  
T Heiskanen ◽  
L Peltonen
Keyword(s):  
Molecular Mass ◽  
Reversed Phase ◽  
Alpha Subunit ◽  
Cross Linking ◽  
Terminal Sequence ◽  
Α Subunit ◽  
Sds Page ◽  
Specific Antisera ◽  
Purification Scheme ◽  
Chemical Cross Linking

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.

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.

scholarly journals The adipocyte Goα-immunoreactive polypeptide is different from the α subunit of the brain Go protein

1989 ◽  
Vol 260 (1) ◽  
pp. 307-310 ◽  
Author(s):  
B Rouot ◽  
J Carrette ◽  
M Lafontan ◽  
P Lan Tran ◽  
J A Fehrentz ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Molecular Mass ◽  
G Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mass Range ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Positive Immunoreactivity ◽  
Rat Adipose Tissue ◽  
The Brain

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.

Molecular characterization of a G protein α-subunit-encoding gene fromMucor circinelloides

2006 ◽  
Vol 52 (7) ◽  
pp. 627-635 ◽  
Author(s):  
Victor Meza-Carmen ◽  
Jesús García-Soto ◽  
Laura Ongay-Larios ◽  
Roberto Coria ◽  
Mario Pedraza-Reyes ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Molecular Mass ◽  
Pertussis Toxin ◽  
Mucor Circinelloides ◽  
Dimorphic Fungus ◽  
Α Subunit ◽  
Calculated Molecular Mass ◽  
C Terminus ◽  
Sds Page ◽  
Genes Encoding

Genes encoding the Gα subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Gα subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%–67% identity with Gα subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast–mycelium and mycelium–yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Gα subunits of the Gαi/o/tsubfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS–PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Gαi/o/t. Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Gα subunit Gpa3 is expressed constitutively during differentiation.Key words: Gα-subunit-encoding genes, Mucor circinelloides, Gpa3 recombinant protein.

scholarly journals The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

1989 ◽  
Vol 261 (3) ◽  
pp. 841-845 ◽  
Author(s):  
L Molina y Vedia ◽  
R D Nolan ◽  
E G Lapetina
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Present Evidence ◽  
Adp Ribosylation ◽  
Specific Antisera ◽  
Membrane Bound ◽  
Gs Alpha ◽  
Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

scholarly journals The interaction between subunits in the tubulin dimer

1985 ◽  
Vol 230 (2) ◽  
pp. 551-556 ◽  
Author(s):  
L Serrano ◽  
J Avila
Keyword(s):  
Formic Acid ◽  
Limited Proteolysis ◽  
Trypsin Digestion ◽  
Cross Linking ◽  
Β Subunit ◽  
Α Subunit ◽  
Tubulin Dimer ◽  
Cross Linker ◽  
Chemical Cross Linking ◽  
Β Tubulin

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between α- and β-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the α-subunit into two fragments, whereas chymotrypsin cleaved the β-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the α-tubulin N-terminal fragment with β-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.

scholarly journals Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays

2019 ◽  
Author(s):  
Andrew Norris ◽  
Florian Busch ◽  
Michael Schupfner ◽  
Reinhard Sterner ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Quaternary Structure ◽  
Early Time ◽  
Cross Linking ◽  
Tryptophan Synthase ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Primary And Secondary Metabolism ◽  
Chemical Cross Linking

The manuscript describes the use of chemical cross-linking/mass spectrometry and mutagenesis to investigate the dimeric interface of the tryptophan synthase α-subunit homolog, BX1. This work indicates that BX1 homodimerization might have served as a mechanism to exclude an interaction with the tryptophan synthase β-subunit, TrpB, at an early time in evolution, thereby eliminating cross-talk between primary and secondary metabolism. This work would be of interest to mass spectrometrists and structural biologist as it presents a workflow to determine the physiological protein-protein interactions within crystal structures using chemical cross-linking/mass spectrometry and mutagenesis as complementary structural biology techniques, thereby eliminating ambiguity and potential mis-assignments due to the presence of additional (artificial) protein contacts formed during the crystallization process.

scholarly journals A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8

1990 ◽  
Vol 271 (3) ◽  
pp. 797-801 ◽  
Author(s):  
B C Beaubien ◽  
P D Collins ◽  
P J Jose ◽  
N F Totty ◽  
J Hsuan ◽  
...  
Keyword(s):  
Peritoneal Cavity ◽  
Inflammatory Reaction ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Interleukin 8 ◽  
Terminal Sequence ◽  
Rabbit Skin ◽  
Sds Page

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.

scholarly journals Probing actin polymerization by intermolecular cross-linking.

1988 ◽  
Vol 106 (3) ◽  
pp. 785-796 ◽  
Author(s):  
R Millonig ◽  
H Salvo ◽  
U Aebi
Keyword(s):  
Molecular Mass ◽  
Actin Filament ◽  
Cell Structure ◽  
Apparent Molecular Mass ◽  
Polymerization Reaction ◽  
Cross Linking ◽  
Actin Monomer ◽  
Oligomeric State ◽  
Sds Page ◽  
Filament Bundles

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.

scholarly journals Formation of salivary-mucosal pellicle: the role of transglutaminase

1992 ◽  
Vol 284 (2) ◽  
pp. 557-564 ◽  
Author(s):  
S D Bradway ◽  
E J Bergey ◽  
F A Scannapieco ◽  
N Ramasubbu ◽  
S Zawacki ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Molecular Mass ◽  
Cross Linking ◽  
Buccal Epithelial Cell ◽  
Sds Page ◽  
Buccal Epithelial Cells ◽  
Cystatin Sn

The present investigation was carried out to identify salivary components of mucosal pellicles in vivo and explore further the mechanism of interaction between salivary molecules and buccal epithelial cells. By using specific antisera and immunoprotein blotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, salivary cystatins and proline-rich proteins were detected within mucosal pellicle in vivo. In addition, the data indicated that the mucins and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. The role of buccal epithelial cell transglutaminase in these interactions was further studied by utilizing purified iodinated amylase, neutral cystatin SN and acidic proline-rich proteins 1 and 3 (APRP1 and 3). After incubation with buccal epithelial cells in vitro 125I-labelled APRPs appeared to undergo a greater degree of cross-linking than 125I-labelled cystatin SN, as determined by SDS/PAGE/autoradiography. Amylase did not appear to be cross-linked at all. Recovery of 125I-labelled APRPs and 125I-labelled cystatin SN with epithelial cell envelopes after repeated extraction suggested that both molecules were cross-linked to envelope proteins, but that 125I-labelled APRPs were cross-linked to a greater degree than 125I-labelled cystatin SN. Cross-linking in buccal epithelial cell preparations was inhibited by an excess of methylamine hydrochloride, a transglutaminase substrate. In a further assessment of amylase, cystatin and APRPs as transglutaminase substrates, only APRP3 and a partially purified preparation of APRPs acted as an amine acceptor for the cross-linking of [14C]methylamine by purified transglutaminase, as determined by SDS/PAGE/fluorography. This reaction was completely inhibited by excess EDTA. The combined data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to buccal epithelial cell surfaces, and that, within this group, selected components are enzymically cross-linked by an epithelial transglutaminase and/or proteolytically cleaved into smaller fragments.

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