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1.
Human pituitary thyrotropin: isolation and chemical characterization of its subunits 总被引:1,自引:0,他引:1
The subunits of human pituitary thyrotropin have been separated and purified by countercurrent distribution and exclusion chromatography. The NH2-terminal sequence of the β subunit is identical to that of the β subunit of bovine thyrotropin. However, amino acid composition and peptide map of tryptic and chymotryptic digests as well as compositions of tryptic and chymotryptic peptides suggest that the amino acid sequence of the α subunit is identical to that of the α subunit of human interstitial cell stimulating hormone. 相似文献
2.
Taruna Arora Nidhi Vashistha Rajesh Chaudhary Kambadur Muralidhar 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(28):2679-2684
The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar–dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue. 相似文献
3.
M R Sairam 《Archives of biochemistry and biophysics》1979,194(1):71-78
The α and β subunits of highly potent ovine follitropin have been isolated by dissociation in 8 m urea, pH 7.5, and chromatography on DEAE-Sephadex A25. The isolated subunits display microheterogeneity on polyacrylamide gel electrophoresis and have very low activity in follitropin-specific radioreceptor and radioimmunoassays. The tryptophan fluorescence spectra of native follitropin and the isolated β subunit are different. The recombinant of follitropin α + β subunit had the same activity as the native hormone in the radioimmunoassay, but its activity in the radioreceptor and in vivo bioassay was about 65% of the intact hormone. Substitution of the follitropin α by ovine lutropin α subunit (prepared by a method not involving urea) to form the recombinant restored full activity in all the three assays investigated. The formation of recombined hormone proceeds at a rapid rate and is almost complete by 6 h. The α and β subunits of ovine follitropin differ from each other in amino acid composition. No significant differences were apparent in their carbohydrate composition. The amino acid composition of the ovine follitropin α and lutropin α subunits are very similar. The oxidized α subunit has phenylalanine at its NH2-terminus while aspartic acid is present at this position in the oxidized β subunit. 相似文献
4.
Garcia-Caballero A Ishmael SS Dang Y Gillie D Bond JS Milgram SL Stutts MJ 《Channels (Austin, Tex.)》2011,5(1):14-22
The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion. 相似文献
5.
The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F1 portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pHi (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pHi of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pHi homoeostasis. 相似文献
6.
Helena Przywarska-Boniecka Lilianna Trynda Teresa Kościukiewicz 《Inorganica chimica acta》1984,93(4):147-152
α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex. 相似文献
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9.
The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding. 相似文献
10.
Mary Ashley Rimmer Owen W. Nadeau Jianyi Yang Antonio Artigues Yang Zhang Gerald M. Carlson 《Protein science : a publication of the Protein Society》2018,27(2):472-484
Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4, and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full‐length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen‐deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N‐terminal glycoside hydrolase domain and a large C‐terminal importin α/β‐like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen‐deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter‐subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low‐exchanging region in the C‐terminus of α that is known to bind the regulatory domain of the catalytic γ subunit. 相似文献
11.
L. L. Isom 《Journal of neurochemistry》2002,81(Z1):68-68
Voltage‐gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore‐forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage‐gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore‐forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin. 相似文献
12.
Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis. 相似文献
13.
The native Ras farnesyltransferase heterodimer (αβ) and a heterodimer with a truncated α subunit (α′β) were overproduced at a high level and in a soluble form inEscherichia coli.The α, α′, and β subunits were synthesized from individual plasmid vectors under the control of bacteriophage T7 promoters. Although each subunit could be expressed at a high level by itself, when either the α or α′ and the β plasmid were present in cells at the same time, the α and α′ subunits were preferentially expressed to such a degree that little or none of the β subunit accumulated. A satisfactory balance between both combinations of subunits (αβ and α′β) was achieved by making incremental adjustments in the copy number of the β-encoding plasmid. As the copy number of the β plasmid increased, so did the ratio of β:α or β:α′, but there was little difference in the total amount of recombinant protein (α + β or α′ + β) that was produced. This may be a generally useful method for balancing the production of two recombinant polypeptides inE. coli.A noteworthy advantage of this approach is that it can be undertaken without first determining the cause of the imbalance. 相似文献
14.
F D Ledley B R Mullin G Lee S M Aloj P H Fishman L T Hunt M O Dayhoff L D Kohn 《Biochemical and biophysical research communications》1976,69(4):852-859
The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A1 chain believed to be responsible for adenylate cyclase activation. 相似文献
15.
Reconstitution of ATPase from the isolated subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3 总被引:2,自引:0,他引:2
M Futai H Kanazawa K Takeda Y Kagawa 《Biochemical and biophysical research communications》1980,96(1):227-234
ATPase was reconstituted from mixtures of isolated subunits of coupling factor, F1 ATPase of E. coli (EF1) and thermophilic bacterium PS3 (TF1); ability to hydrolyze ATP was attained from the combination of α and β subunits from EF1 and γ subunit from TF1, α and β from TF1 and γ from EF1, and α and γ from EF1 and β from TF1. The β subunit of TF1 also could complement the EF1 from an E. coli mutant defective in this subunit. This is the first demonstration of interspecies in vitro recombination of ATPase activity from isolated subunits. 相似文献
16.
C.N. Rao I.M.K. Margulies R.H. Goldfarb J.A. Madri D.T. Woodley L.A. Liotta 《Archives of biochemistry and biophysics》1982,217(1):65-71
Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) Mr on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 Mr whereas plasmin produced two fragments of 180,000 and 140,000 Mr. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 Mr) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule. 相似文献
17.
H Papkoff 《Biochemical and biophysical research communications》1974,58(2):397-404
The two subunits (α and β) of pregnant mare serum gonadotropin have been dissociated and partially characterized. Recombination of the biologically inactive subunits results in the restoration of both the follicle stimulating and leuteinizing activities of pregnant mare serum gonadotropin. In addition, the α subunit of pregnant mare serum gonadotropin can be combined with the β subunit of either ovine luteinizing hormone, human chorionic gonadotropin, or follicle stimulating hormone with generation of the specific activity expected of the β subunit. 相似文献
18.
Adenine nucleotide binding sites in normal and mutant adenosine triphosphatases of Escherichia coli 总被引:2,自引:0,他引:2
Three types of assays were used to characterize adenine nucleotide binding sites on the Ca2+, Mg2+-activated ATPase of normal Escherichia coli and its unc A 401 and unc D 412 mutants. ADP was bound mainly at a single site in normal and mutant ATPase. In the absence of divalent cations ATP was bound at a single high-affinity and three low-affinity sites in normal and unc D ATPases. The 2′,3′-dialdehyde (oADP) obtained by periodate oxidation of ADP reacted with both low- and high-affinity sites whereas oATP was bound primarily at a low-affinity site. Two types of adenine nucleotide binding sites, a high-affinity site reacting with ATP and ADP and a low-affinity site for ATP, were detected by the effects of these nucleotides on the fluorescence of the aurovertin D-ATPase complex. This high-affinity site(s) was present in normal and mutant ATPases. However, the fluorescence response at both high- and low-affinity sites was modified in the unc D ATPase as a consequence of the abnormal β subunit in this enzyme. Normal fluorescence responses were not induced by the binding of oADP or oATP to the ATPases. ATP was bound at a single site on isolated α subunits of the enzyme. Since this site was not detected in the unc A ATPase, it is unlikely to be the high-affinity site detected in the intact enzyme or the binding site for the endogenous tightly bound adenine nucleotides found in the purified ATPase. It is more probable that the site detected on the isolated α subunit from the normal enzyme is that which binds oADP since this site was absent in the unc A ATPase. Pretreatment of the normal ATPase with either N, N′-dicyclohexyl-carbodiimide (DCCD) or with 4-chloro-7-nitrobenzofurazan (NbfCl), reagents which inhibit ATPase activity by reacting with a β subunit, affected binding of oADP to α subunit(s) but had less effect with oATP. Inhibition of oADP binding could be due to conformational changes induced in the α subunit by the reaction of DCCD and NbfCl with a β subunit, or to steric reasons. If the latter hypothesis is correct, the active site of the ATPase would be at the interface between α and β subunits of the enzyme. 相似文献
19.
Nucleotide sequence of the genes coding for alpha, beta and gamma subunits of the proton-translocating ATPase of Escherichia coli 总被引:12,自引:0,他引:12
H Kanazawa T Kayano K Mabuchi M Futai 《Biochemical and biophysical research communications》1981,103(2):604-612
A nucleotide sequence of 2328 base pairs comprising a portion of the gene cluster for the proton-translocating ATPase of was determined. The sequence covers most of the gene for α subunit, the entire gene for γ subunit and the amino terminal portion of the gene for β subunit, along with the flanking regions of these genes. The amino acid sequences of these subunits deduced from the DNA sequences indicate that the α and γ subunits have 513 and 287 amino acid residues, respectively. A possible secondary structure for each subunit was estimated from the inferred primary structure. The intercistronic regions between the genes for α and γ and between γ and β are 49 and 26 base pairs, respectively. The significance of codon usage in these genes is discussed in correlation with their expression. 相似文献