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 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 Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1α when constrained within an α2A-adrenoceptor-Gi1α fusion protein

1997 ◽  
Vol 325 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Alan WISE ◽  
I. Craig CARR ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Pertussis Toxin ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Fusion Construct ◽  
High Affinity ◽  
C Terminus ◽  
Adrenoceptor Agonist ◽  
Stimulation Of

A fusion protein was generated between the porcine α2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351 → Gly) variant of the α subunit of Gi1α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min-1 for Gi1α when the α2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37±0.07 μM. Co-expression of excess β1γ2 along with the α2A-adrenoceptor-Gi1α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

scholarly journals Group B streptococci inactivate complement component C5a by enzymic cleavage at the C-terminus

1991 ◽  
Vol 273 (3) ◽  
pp. 635-640 ◽  
Author(s):  
J F Bohnsack ◽  
K W Mollison ◽  
A M Buko ◽  
J C Ashworth ◽  
H R Hill
Keyword(s):  
Molecular Mass ◽  
Critical Role ◽  
Polymorphonuclear Leucocytes ◽  
Group B Streptococci ◽  
Plasma Desorption ◽  
C Terminus ◽  
Sds Page ◽  
Human Pmns ◽  
Group B

Incubation of recombinant human C5a (rC5a) with the 7360 strain of group B streptococci (GBS) destroyed the ability of rC5a to stimulate chemotaxis or adherence of purified human polymorphonuclear leucocytes (PMNs). Treatment of 125I-labelled rC5a with GBS 7360 correspondingly decreased rC5a binding to human PMNs. This also resulted in an approx. 600 Da decrease in the molecular mass of rC5a as determined by SDS/PAGE. Incubation of rC5a with the GBS strain GW, which only minimally altered the ability of rC5a to activate human PMNs, did not affect rC5a binding to PMNs and did not alter the molecular mass of rC5a on SDS/PAGE. Plasma-desorption m.s. of rC5a inactivated by GBS 7360 showed that the GBS cleaved the rC5a between histidine-67 and lysine-68 near the C-terminus of rC5a. This mechanism of inactivation of C5a by proteolytic cleavage at the C-terminus of C5a is consistent with the known critical role of the C-terminus in C5a activation of human PMNs. This C5a-cleaving proteinase activity may contribute to the pathophysiology of GBS infections.

scholarly journals TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family

2007 ◽  
Vol 403 (3) ◽  
pp. 583-591 ◽  
Author(s):  
Ellen Fierens ◽  
Sigrid Rombouts ◽  
Kurt Gebruers ◽  
Hans Goesaert ◽  
Kristof Brijs ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Amino Acid ◽  
Molecular Mass ◽  
Glycoside Hydrolase ◽  
Enzyme Inhibitor ◽  
Glycoside Hydrolase Family ◽  
Calculated Molecular Mass ◽  
Sds Page ◽  
Xylanase Inhibitor ◽  
Hydrolase Family

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI≥9.3 in isoelectric focusing) protein with a molecular mass of approx. 18–kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6–kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21–kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a Ki of approx. 60–nM. Except for zeamatin, an α-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

scholarly journals Genes encoding ribonucleoside hydrolase 1 and 2 from Corynebacterium ammoniagenes

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1169-1177 ◽  
Author(s):  
Hyun-Soo Kim ◽  
Jin-Ho Lee ◽  
Won-Sik Lee ◽  
Won-Gi Bang
Keyword(s):  
Molecular Mass ◽  
Minimal Medium ◽  
Calculated Molecular Mass ◽  
Corynebacterium Ammoniagenes ◽  
E Coli ◽  
Pyrimidine Nucleosides ◽  
Genes Encoding ◽  
Salvage Pathways ◽  
Nucleoside Hydrolases ◽  
Hydrolysis Of

Two kinds of nucleoside hydrolases (NHs) encoded by rih1 and rih2 were cloned from Corynebacterium ammoniagenes using deoD- and gsk-defective Escherichia coli. Sequence analysis revealed that NH 1 was a protein of 337 aa with a deduced molecular mass of 35 892 Da, whereas NH 2 consisted of 308 aa with a calculated molecular mass of 32 310 Da. Experiments with crude extracts of IPTG-induced E. coli CGSC 6885(pTNU23) and 6885(pTNI12) indicated that the Rih1 enzyme could catalyse the hydrolysis of uridine and cytidine and showed pyrimidine-specific ribonucleoside hydrolase activity. Rih2 was able to hydrolyse both purine and pyrimidine ribonucleosides with the following order of activity – inosine>adenosine>uridine>guanosine>xanthosine>cytidine – and was classified in the non-specific NHs family. rih1 and rih2 deletion mutants displayed a decrease in cell growth on minimal medium supplemented with pyrimidine and purine/pyrimidine nucleosides, respectively, compared with the wild-type strain. Growth of each mutant was substantially complemented by introducing rih1 and rih2, respectively. Furthermore, disruption of both rih1 and rih2 led to the inability of the mutant to utilize purine and pyrimidine nucleosides as sole carbon source on minimal medium. These results indicated that rih1 and rih2 play major roles in the salvage pathways of nucleosides in this micro-organism.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Biosynthesis of the Cyanobacterial Light-Harvesting Polypeptide Phycoerythrocyanin Holo-α Subunit in a Heterologous Host

2002 ◽  
Vol 184 (17) ◽  
pp. 4666-4671 ◽  
Author(s):  
Aaron J. Tooley ◽  
Alexander N. Glazer
Keyword(s):  
Ternary Complexes ◽  
Covalent Attachment ◽  
Heme Oxygenase 1 ◽  
Α Subunit ◽  
Ionization Time ◽  
E Coli ◽  
Metal Affinity ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-α subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter. Genes for the apo-phycoerythrocyanin α subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid. Upon induction, recombinant E. coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria. About two-thirds of the apo-PecA was converted to holo-PecA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks pecE and pecF. By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF. The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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