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1.
L-alpha-Hydroxyacid oxidase isozymes from rat liver (A isozyme) and kidney (B isozyme) have been isolated in a high state of purity with specific activities of 61 and 14.7 microkatals per gram protein respectively. The subunit molecular weights determined by sodium dodecylsulphate polyacrylamide gel electrophoresis were 40000 +/- 3000; the mouse A and B isozymes were also partially purified and their subunit molecular weights shown to be 37000. 相似文献
2.
Resonance energy transfer between the active sites of myocardial-type creatine kinase (isozyme MB) 总被引:1,自引:0,他引:1
S H Grossman 《Biochemistry》1983,22(23):5369-5375
The single reactive sulfhydryl group, located in the active site of each subunit of dimeric creatine kinase from rabbit muscle (isozyme MM), was selectively labeled with 3-(4-maleimidylphenyl)-7-(diethylamino)-4-methylcoumarin (CPM). Isozyme BB, purified to homogeneity from rabbit brain, was conjugated with the sulfhydryl-specific reagent 5'-(iodoacetamido)fluorescein (5'-IAF). Spectral analyses demonstrated that 1.8 mol of CPM and 1.9 mol of 5'-IAF had reacted per mol of protein. Labeled isozymes were combined, denatured in 8 M urea, and renatured by dialysis, producing the parent labeled homodimers and forming the heterolabeled hybrid dimer, creatine kinase MB. Similar hybridizations were performed to prepare singly labeled hybrids, starting with labeled and unlabeled homodimers. The hybrid isozymes were isolated by ion-exchange chromatography, and spectral analyses of singly labeled heterodimers revealed overlap between the absorption spectrum of MB labeled with acetamidofluorescein on the B subunit and the corrected fluorescence emission spectrum of MB labeled with CPM on the M subunit. Analyses included evaluation of the quantum yield of the CPM-labeled hybrid, estimation of the range of the orientation factor K2 from fluorescence polarization and anisotropy studies, and determination of J, the spectral overlap integral for the fluorescence donor (CPM-labeled MB) and acceptor (acetamidofluorescein-labeled MB). Results of these experiments permitted an estimation of R0, the distance between the donor and the acceptor at which energy transfer is 50% efficient. Comparison of the relative fluorescence of the donor in the presence (heterolabeled hybrid) and absence (hybrid conjugated with CPM on the M subunit) of the acceptor or determination of the normalized sensitization of the acceptor fluorescence led to an evaluation of the transfer efficiency and the actual transfer distance of between 27 and 52 A.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins. 总被引:1,自引:0,他引:1
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Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit. 相似文献
4.
E G Lufkin O D Taunton F B Stifel F D Hofeldt M R Wrensch L Hagler R H Herman 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1975,150(2):410-413
Antisera against rat liver aspartate aminotransferase (EC 2.6.1.1) isozymes were used to study the activity and immunologic pattern of these isozymes in the livers of the rat, mouse, hamster, gerbil and in Ehrlich ascites cells. A double immunodiffusion precipitin test and immunoelectrophoresis showed that, except for the gerbil, there was a pattern of identity of AAT isozymes in the presence of either the antianionic or the anticationic antisera. Although gerbil AAT isozymes are immunochemically different from those of the other rodents studied, they were inactivated by the respective antiserum in a manner similar to that observed with the other species. This may suggest that antigenic determinants at the catalytic site of each of the liver aspartate aminotransferase isozymes are least likely to change throughout the evolutionary process. 相似文献
5.
Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase : Purification, N-Terminal Sequencing, and Antibody Production 总被引:7,自引:6,他引:1
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In black cherry (Prunus serotina Ehrh.) seed homogenates, amygdalin hydrolase (AH) participates with prunasin hydrolase (PH) and mandelonitrile lyase in the sequential degradation of (R)-amygdalin to HCN, benzaldehyde, and glucose. Four isozymes of AH (designated AH I, I′, II, II′) were purified from mature cherry seeds by concanavalin A-Sepharose 4B chromatography, ion-exchange chromatography, and chromatofocusing. All isozymes were monomeric glycoproteins with native molecular masses of 52 kD. They showed similar kinetic properties (pH optima, Km, Vmax) but differed in their isoelectric points and N-terminal amino acid sequences. Analytical isoelectric focusing revealed the presence of subisozymes of each isozyme. The relative abundance of these isozymes and/or subisozymes varied from seed to seed. Three isozymes of PH (designated PH I, IIa, and IIb) were purified to apparent homogeneity by affinity, ion-exchange, and hydroxyapatite chromatography and by nondenaturing polyacrylamide gel electrophoresis. PH I and PH IIb are 68-kD monomeric glycoproteins, whereas PH IIa is dimeric (140 kD). The N-terminal sequences of all PH and AH isozymes showed considerable similarity. Polyclonal antisera raised in rabbits against deglycosylated AH I or a mixture of the three deglycosylated PH isozymes were not monospecific as judged by immunoblotting analysis, but also cross-reacted with the opposing glucosidase. Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen. 相似文献
6.
F Berrocal J Carreras 《Comparative biochemistry and physiology. B, Comparative biochemistry》1987,86(3):547-554
Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast. 相似文献
7.
Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases 总被引:5,自引:0,他引:5
C P Tu A Matsushima N Q Li D M Rhoads K Srikumar A P Reddy C C Reddy 《The Journal of biological chemistry》1986,261(20):9540-9545
The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs. 相似文献
8.
The major internal protein of a rat type C virus pseudotype of murine sarcoma virus, MSV(RaLV), was purified by isoelectric focusing (pI = 8.6) and used to prepare antibody in guinea pigs. The protein was identified by its reaction with antisera reactive with the mammalian type C virus group-specific (gs) antigenic determinant, gs-3. The guinea pig antisera mainly contained species-specific (gs-1) antibody for reactions in gel diffusion with other type C viruses were limited to those of rat origin, whereas in complement fixation tests heterologous reactions could be eliminated by use of appropriate antiserum concentrations without affecting homologous reactions. Guinea pig antisera against mouse, hamster, or cat gs-1 determinants did not react with MSV(RaLV) purified gs protein or with any of several other rat type C viruses. 相似文献
9.
A sensitive and convenient method for the quantitative measurement of human alcohol dehydrogenase (ADH) isozymes based on enzyme-linked immunosorbent assay has been devised. The procedure was optimized with respect to antigen coating density, antiserum dilution, and incubation times with rabbit antisera raised against beta 1 beta 1-ADH to achieve a limit of sensitivity of 1 ng/ml for this isozyme when purified. Using the optimal conditions established, quantitative measurement of alpha beta 1, alpha gamma 1, beta 1 gamma 1, pi, and chi-ADH were obtained with antisera raised in rabbits toward these individual isozymes. The incorporation into the procedure of thimerosal (ethyl(4-mercaptobenzoato-S)mercury) or other sulfhydryl specific reagents improved the soluble phase antiserum avidity for all ADH isozymes, thereby increasing the sensitivity. Thimerosal is an absolute requirement for chi-ADH antigen-antibody binding. The polyclonal rabbit antisera elicited by the individual isozymes of the three classes of ADH exhibit a high degree of isozyme class specificity. Cross-reactivity of the antibodies with the beta 1 beta 1, alpha gamma 1, alpha gamma 2, alpha beta 1, beta 1 gamma 1, beta 1 gamma 2, pi and chi isozymes were evaluated. Antisera against the class I isozymes beta 1 beta 1 and beta 1 gamma 1 cross-react with all class I isozymes and with pi-ADH. Antibodies against pi and chi-ADH are selective and specific only for their respective antigens. Neither one cross-reacts with any class I isozyme. Conformational effects resulting from subunit interactions likely account for differences in cross-immunoreactivity between the closely homologous class I isozymes. 相似文献
10.
V N Malakhov V A Tishchenko I I Efron E A Chukhri? V A Isachenkov 《Biokhimii?a (Moscow, Russia)》1977,42(7):1221-1231
Properties of human creatine kinase isoenzymes (MM, MB and BB) are investigated. The most pronounced differences in properties of these isoenzymes are found under their urea inactivation, heat denaturation and the inhibition by rabbit antisera to isoenzymes. Differences in values of the Mikhaelis constant and substrate and pH dependencies are much less pronounced. The presence of ADP stabilizes creatine kinase isoenzymes under conditions of urea and heat inactivation. Properties of hybrid MB isoenzymes are found to be intermediate with respect to MM and BB isoenzymes. A mode of the interaction of M and B subunits in dimeric molecules of creatine kinase isoenzymes is discussed. 相似文献
11.
Hughes MJ Wilson R Moore JC Lane JD Dobson RJ Muckett P Younes Z Pribul P Topping A Feldman RG Santangelo JD 《FEMS microbiology letters》2003,222(2):263-271
Using an alkaline phosphatase-based genetic screening method, we identified a number of proteins that are potentially located on the outer surface of Group B streptococcus (Streptococcus agalactiae). In an enzyme-linked immunosorbent assay, antisera raised against two of the proteins, the streptococcal yutD homologue and a subunit of an ABC transporter, recognised clinically important serotypes of Group B streptococcus. In a neonatal rat model, purified IgG from the sera conferred significant levels of protection against a lethal challenge infection. The proteins identified show potential as protein subunit candidates for vaccines against Group B streptococcal disease in neonates. 相似文献
12.
谷胱甘肽转移酶(EC 2,5,1,18 Glutathione S-transferases简称GSTs)是一组具有多种生理功能的蛋白质。我们通过105,000×g超速离心,s—已基—谷胱甘肽—Sepharose-6B亲和层析柱和DEAE52纤维柱或CM52纤维柱将人肝粗匀浆纯化为电泳纯的GSTs同工酶。经系和层析柱后GSTs比活比粗匀浆上清液提高54倍,回收率近60%。通过DE52柱将人肝GSTs分离为7个同工酶组分,分别称为c_(DE),A_1,A_2,A_3,A_4,A_5和A_6,经等电聚焦电泳和SDS-pAGE电泳鉴定,其等电点依次为8.60,7.05,6.70,6:60,6.55,6.45和6.4。经CM52柱后得到5个不同的同工酶组分,分别定名为A_(CM),c_1,c_2,c_3和c_4等电点各自为 6.30,7.00,8.50,8.55和8.60。阳离子同工酶(即c_(DE),C_1,C_2,C_3和C_4)的分子量在23,500—24,000道尔顿,阴离子同工酶(A_(CM),A_1-A_6)约为25,000道尔顿。并将亲和层析柱后样品,阳离子同工酶C_(DE)和阴离子同工酶A_(CM)作为抗原,得到兔抗人肝GSTs相应同工酶的抗血清,其抗血清效价经免疫双扩散法测定分别为1:96,1:64,1:16。并对人肝GSTs进行氨基酸组份的测定。 相似文献
13.
The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase. 相似文献
14.
Dissociation, reassociation, and purification of plastid and cytosolic phosphoglucose isomerase isozymes 总被引:7,自引:4,他引:3
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The plastid and cytosolic isozymes of the dimeric enzyme phosphoglucose isomerase (EC 5.3.1.9) from spinach (Spinacia oleracea) and cauliflower (Brassica oleracea) were purified to apparent homogeneity. The isozymes from sunflower (Helianthus annuus) and Clarkia xantiana were partially purified. When subunits from two electrophoretically distinguishable cytosolic isozymes, either from the same or from different species, were dissociated and allowed to reassociate in each other's presence, an active hybrid enzyme, consisting of one subunit of each type, was formed in addition to the two original homodimers. Active hybrid enzymes were also formed by dissociation and reassociation of plastid isozymes. Hybrid molecules were not produced between the plastid and cytosolic subunits, suggesting that they are not able to bind with each other. Additional differences between the plastid and cytosolic isozymes are described. 相似文献
15.
Nature of the rat brain 6-phosphofructo-1-kinase isozymes 总被引:1,自引:0,他引:1
The complex nature of the brain 6-phosphofructo-1-kinase isozymes was examined by elution with a discontinuous gradient from QAE (quaternary aminoethyl)-Sephadex. In the first wash (150 mM NaCl), where the rat muscle 6-phosphofructo-1-kinase isozyme (M4) eluted, about 40% of the total brain 6-phosphofructo-1-kinase activity washed through without exhibiting a sharp peak. In the second elution (300 mM NaCl), the remaining activity eluted in a sharp peak that preceded where the major rat liver 6-phosphofructo-1-kinase isozyme (L4) eluted. Enzyme activity in brain extracts or purified brain isozymes was titrated above 90% with M4 anti-IgG and 20% with L4 anti-IgG. A purification procedure was developed which resulted in a recovery of 70 to 80% of the original enzyme activity in brain 100,000 X g supernatant fluids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on slab gels and detection by silver staining indicated that three components were present with apparent molecular weights of 87,500, 85,000, and 80,000. The 85,000- and 80,000-dalton components corresponded to the subunits of M4 and L4, respectively. The third component (C type) was thought to be an actual subunit since it exhibited the highest molecular weight and was present in an exhaustively washed immunoprecipitate of the purified brain isozymes. From 10 different purifications of the brain enzyme, the subunit distributions of the liver, muscle, and C-type subunit were 1.4 +/- 0.2, 4.9 +/- 0.5, and 3.9 +/- 0.3, respectively. A comparison of the kinetic properties of purified liver, muscle, and brain isozymes clearly demonstrated that all three preparations had quantitatively different regulatory properties. All three subunits were present in different regions of the brain, and region-specific changes in total activity and the relative amounts of each subunit were observed. This study suggests that brain 6-phosphofructo-1-kinase is a complex mixture of homotetramers and hybrids which are composed of different amounts of the three subunits. 相似文献
16.
Two isozymes of the Na,K-ATPase have distinct antigenic determinants 总被引:11,自引:0,他引:11
Two isozymes of the Na,K-ATPase were purified from rat renal medulla and rat brainstem axolemma, and antisera were raised in rabbits. When antibody titers were measured, two sera showed specificity for either the kidney or axolemma Na,K-ATPases and had limited cross-reactivity which could be removed by cross-adsorption. In blots of polyacrylamide gels, these sera reacted with only the alpha or alpha (+) Na,K-ATPase catalytic subunits, while they cross-reacted with both types of beta subunits. Two other sera each recognized both alpha and alpha (+), indicating that the catalytic subunit isozymes have additional shared antigenic determinants. A comparison of the Na,K-ATPases from the brains of different vertebrate species indicates that birds and fish differ from mammals and amphibians in the manifestation of Na,K-ATPases isozymes. Neither neuraminidase nor endoglycosidase F treatment eliminated specific antibody reaction or affected the electrophoretic mobilities of the alpha and alpha (+) subunits, although endoglycosidase F increased the mobilities of the two types of beta subunits to similar final apparent molecular weights. Blots of the peptide fragments produced by incomplete papain and trypsin digests of the alpha and alpha (+) subunits were stained with the specific sera, and the patterns of immunoreactive fragments were found to be markedly different. The results suggest that the antigenic differences reside in differences in the primary protein sequences of the two isozymes. 相似文献
17.
J Carreras J Bosch J Mezquita 《Comparative biochemistry and physiology. B, Comparative biochemistry》1982,71(1):57-63
1. The three phosphoglycerate mutase isozymes from mammals (types M, B and MB isozymes) differ in their sensitivity to the - SH group reagents. 2. Rabbit muscle phosphoglycerate mutase (type M isozyme) is reversibly inactivated by tetrathionate, rho-chloromercuribenzoate and Hg2+. 3. Titration with rho-chloromercuribenzoate shows the existence of two sulfhydryl groups per enzyme subunit, the modification of which produces a progressive decline in enzyme activity. 4. The apparent Km values for substrate and cofactor are not affected by tetrathionate treatment. 5. Phosphoglycerate mutase inactivated by tetrathionate and by rho-chloromercuribenzoate is unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P2, and is not protected by the cofactor against heating. 6. Glycerate-2,3-P2 protects against tetrathionate treatment, but fails to protect against Hg2+ and rho-chloromercuribenzoate inactivation. 相似文献
18.
G. Egea J. M. Ureña X. Graña J. Marsal J. Carreras F. Climent 《Histochemistry and cell biology》1992,97(3):269-275
Summary We have previously reported (Ureña et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.Abbreviations PGAM
phosphoglycerate mutase
- PGAM-M(M)
muscle specific subunit (isozyme) of PGAM
- PGAM-B(B)
brain type subunit (isozyme) of PGAM
- ssDNA
single stranded DNA
- PBS
0.001 M phosphate buffer, pH 7.4, containing 0.15 M NaCl
- kDa
kilodalton 相似文献
19.
Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences 总被引:2,自引:1,他引:1
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We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. 相似文献
20.
Briganti F Pessione E Giunta C Mazzoli R Scozzafava A 《Journal of Protein Chemistry》2000,19(8):709-716
Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR features typical of Fe(III) intradiol dioxygenases. The kinetic properties of the two enzymes such as the specificities toward substituted catechols, the main catalytic parameters, and their behavior in the presence of different kind of inhibitors are, unexpectedly, very similar, in contrast to most of the previously known dioxygenase isozymes. 相似文献