共查询到20条相似文献,搜索用时 31 毫秒
1.
T Kanaji H Ozaki T Takao H Kawajiri H Ide M Motoki Y Shimonishi 《Canadian Metallurgical Quarterly》1993,268(16):11565-11572
The complete amino acid sequence of transglutaminase (EC 2.3.2.13) (TGase), which is produced by a microorganism, Streptoverticillium sp. strain s-8112, and catalyzes the acyl transfer reaction between gamma-carboxyamide groups of glutamine residues in proteins and various primary amines, has been established by a combination of fast atom bombardment mass spectrometry and standard Edman degradation of peptide fragments produced by treatment of the TGase with various proteolytic enzymes and purified by a reversed-phase high performance liquid chromatography. The TGase consists of 331 amino acid residues with a chemical molecular weight of 37,863, in agreement with the observed molecular weight (37,869.2 +/- 8.8) determined from its electrospray ionization mass spectrum. The sequence of the enzyme is very different from those of mammalian TGases represented by guinea pig liver enzyme. The enzyme contains a sole Cys residue, which is essential for its catalytic activity. Hydropathy analysis indicated that the secondary structure of the region around the active site Cys residue is similar to those of mammalian TGases. These results suggest that this microbial protein evolved by a different pathway from that of mammalian TGases and acquired acyl transfer activity during the evolutional process. 相似文献
2.
GTP-binding protein/transglutaminases (tissue transglutaminases or TGases) have been implicated in a variety of cellular processes including retinoic acid (RA)-induced apoptosis. Recently, we have shown that RA activates TGases as reflected by stimulated GTP binding, increased membrane association, and stimulated phosphoinositide lipid turnover. This prompted us to search for cellular proteins that bind TGases in a RA-stimulated manner. In this report, we show that the eukaryotic initiation factor (eIF-5A), a protein that is essential for cell viability, perhaps through effects on protein synthesis and/or RNA export, associates with the TGase in vivo. The interaction between eIF-5A and TGase is specific for the GDP-bound form of the TGase and is not detected when the TGase is pre-loaded with GTP gamma S. The TGase-eIF-5A interaction also is promoted by Ca2+, Mg2+, and RA treatment of HeLa cells. In the presence of retinoic acid, millimolar levels of Ca2+ are no longer required for the TGase-eIF-5A interaction. Nocodazole treatment, which blocks the cell cycle at mitosis (M phase), strongly inhibits the interaction between eIF-5A and cytosolic TGase. The interaction between TGase and eIF-5A and its sensitivity to the nucleotide-occupied state of the TGase provides a potentially interesting connection between RA signaling and protein synthesis and/or RNA trafficking activities. 相似文献
3.
E Tarcsa E Candi T Kartasova WW Idler LN Marekov PM Steinert 《Canadian Metallurgical Quarterly》1998,273(36):23297-23303
The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no alpha or beta structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substrate in vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2. 相似文献
4.
K Sano K Nakanishi N Nakamura M Motoki H Yasueda 《Canadian Metallurgical Quarterly》1996,60(11):1790-1794
We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure. 相似文献
5.
Phospholipase D (EC 3.1.4.4) has been implicated in diverse cellular processes, but its physiological role is not well established in plants. In order to develop immunological and molecular biology approaches to address the problem, we report here the immunological analysis and N-terminal amino acid sequence of a cytosolic phospholipase D from castor bean (Ricinus communis L.). The enzyme was purified to apparent homogeneity from germinating castor bean endosperm. The specific activity of the purified enzyme was enhanced by approximately 670-fold with an overall yield of 4%. Its molecular mass was estimated at 92 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this enzyme was KLVENIEETVGFGKG. Polyclonal antibodies were raised against the purified enzyme. The antibodies inhibited the activity of transphosphatidylation more than that of hydrolysis of phospholipase D. The differential effect on the two activities of this enzyme implies that different active sites on this enzyme may be involved in the two reactions. Immunoblot analyses showed that the amounts of phospholipase D protein relative to the total endosperm proteins increased during the first 5 days of germination. The antibodies cross-reacted to proteins from several tested plant species, and those proteins had molecular masses similar to that of castor bean phospholipase D. These results indicate that the expression of phospholipase D in castor bean changes according to growth stages and that phospholipase D enzymes of different plant species are structurally related. 相似文献
6.
2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases. 相似文献
7.
NADH-dependent glutamate synthase (Nadh-Gogat; EC 1.41.14) was purified 766-fold from the fat body of 5th instar larvae of the silkworm with a final specific activity of 13.8 units/mg protein by a produce including ammonium sulfate fraction, Q-Sepharose HP ion exchange column chromatography, Blue Sepharose FF affinity column chromatography and Superdex 200 HR gel filtration. The purified enzyme yielded a single band corresponding to a molecular mass of 195kDa by SDS-polyacrylamide gel electrophoresis. Molecular mass of the native enzyme was estimated to be 190 kDa by Superdex 200HR gel filtration, suggesting that the enzyme is composed of a monomeric polypeptide. The enzyme showed an absorption spectrum with maximum values at 272, 375, and 435 nm, suggesting the presence of a flavin prosthetic group in the enzyme. The N-terminal amino acid sequence showed a high similarity to those of other GOGATs from plants, yeast and bacteria, but no similarity to other known proteins was detected. The enzyme exhibited a strong specificity to the electron donor and substrates; NADH as electron donor, 2-oxoglutarate as amino acceptor and glutamine as amino donor were essential for the catalytic activity. The optimum pH was around 7.5, at which Km values for 2-oxoglutarate, glutamine and NADH were 17, 220 and 5.7 micro M, respectively. Azaserine, 6-diazo-5-oxonorleucine and p-chloromercuribenzoic acid were strong inhibitors of the activity. These result show that NADH-GOGAT in the silkworm fat body strongly resembles other eukaryotic NADH-GOGATs, suggesting that it plays a significant role in ammonia assimilation in the same manner as the other enzymes. 相似文献
8.
An NADPH-dependent succinic semialdehyde reductase has been purified from bovine brain by several chromatographic procedures. The preparation appeared homogeneous on SDS/PAGE. The enzyme is a monomeric protein with a molecular mass of 28 kDa. A number of properties of the bovine brain enzyme, such as substrate specificity, specific activity, molecular mass, optimum pH, amino acid composition, and kinetic parameters, have been determined and compared with those reported for preparations from other sources. The results indicate that the enzyme isolated from bovine brain in the present study is different from those reported for preparations from other sources. The inhibition kinetic patterns obtained when the products of the reaction or substrate analogs are used as inhibitor of the reaction catalyzed by the enzyme are consistent with an ordered sequential mechanism involving the formation of an intermediate ternary complex and in which NADPH is the first substrate to bind the enzyme. 相似文献
9.
Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced. 相似文献
10.
We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase. 相似文献
11.
DE Garsetti MR Steiner F Holtsberg LE Ozgür RW Egan MA Clark 《Canadian Metallurgical Quarterly》1993,6(1-3):223-232
Lysophospholipases participate in the regulation of the levels of lysophospholipid, compounds with pleiotropic biological effects. Lysophospholipases were purified from a macrophage cell line (WEHI 265.1), a myelocytic leukemia cell line (HL-60) and peripheral blood eosinophils. WEHI 265.1 cells contain three lysophospholipases 28, 27 and 110 kDa as determined by polyacrylamide gel electrophoresis. The 110 kDa lysophospholipase also exhibits phospholipase A2 activity and appears to be identical to a previously described 110 kDa phospholipase A2. Similarly, the HL-60 cells have three lysophospholipases, the largest again a 110 kDa enzyme with phospholipase A2 activity and the smaller are 20 and 21 kDa. The low molecular mass lysophospholipases have distinctive chromatographic properties and amino acid compositions. However, the two low molecular mass enzymes from a given cell type are not radically different, e.g., 15 of the 20 amino acids of the C-terminal sequences of the HL-60 enzymes are identical. A single lysophospholipase, approx. 15 kDa, is a major eosinophil protein. This enzyme is different from those described above. 相似文献
12.
The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases 总被引:1,自引:0,他引:1
E Tarcsa LN Marekov J Andreoli WW Idler E Candi SI Chung PM Steinert 《Canadian Metallurgical Quarterly》1997,272(44):27893-27901
Trichohyalin (THH) is a major structural protein of the inner root sheath cells and medulla layer of the hair follicle and, to a lesser extent, of other specialized epithelia. THH is a high molecular weight insoluble alpha-helix-rich protein that forms rigid structures as a result of postsynthetic modifications by two Ca2+-dependent enzymes, transglutaminases (TGases) (protein cross-linking) and peptidyl-arginine deiminase (conversion of arginines to citrullines with loss of organized structure). The modified THH is thought to serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope. In this paper, we have explored in vitro the order of processing of THH to fulfill these functions, using an expressed truncated, more soluble form THH-8. THH-8 is a complete substrate for three known TGases expressed in epithelia, but the kinetic efficiency with TGase 3 is by far the greatest. Following maximal conversion of its arginines to citrullines, THH-8 is cross-linked even more efficiently by TGase 3, using most glutamines partially and all lysines. In addition, we show that insoluble aggregates of THH-8 or native pig tongue THH can be solubilized following peptidyl-arginine deiminase modification. Together, these data suggest an in vivo model in which THH located in insoluble cytoplasmic droplets is first modified by peptidyl-arginine deiminase which denatures it and makes it more soluble. This renders it available for efficient cross-linking by TGase 3 to form highly cross-linked rigid structures in the cells. This temporal order of reaction is supported by the observation that THH is expressed in hair follicle cells before the TGase 3 enzyme. 相似文献
13.
Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising > 80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated < 5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine. 相似文献
14.
GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. 相似文献
15.
I Lefkovits JR Frey L Kuhn JR Kettman G Béhar C Auffray JP Hoffmann C Coleclough 《Canadian Metallurgical Quarterly》1995,5(1):35-42
beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution. 相似文献
16.
TP Hilbert RJ Boorstein HC Kung PH Bolton D Xing RP Cunningham GW Teebor 《Canadian Metallurgical Quarterly》1996,35(8):2505-2511
We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny. 相似文献
17.
M Ghosh AM Grunden DM Dunn R Weiss MW Adams 《Canadian Metallurgical Quarterly》1998,180(18):4781-4789
Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family. 相似文献
18.
S Sakasegawa I Yoshioka S Koga M Takahashi K Matsumoto H Misaki T Ohshima 《Canadian Metallurgical Quarterly》1998,62(12):2388-2395
A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue. 相似文献
19.
Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a series of hydrophobic affinity column chromatography steps. The activity was enriched 176,000-fold from concentrated urine by only four columns, including octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall recovery was about 20% but only low amounts were obtained due to its low abundance. The estimated final specific activities of several batches were between 1 and 2 mmol/h per mg protein. The final purified fractions were essentially free of other lysosomal enzyme activities. The most pure fractions showed a series of bands between 50 and 53 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have identical N-terminal amino acid sequence. In addition, gel filtration of partially purified GALC after disassociation showed one peak of activity estimated to have a molecular mass near 50 kDa. GALC was also purified from human brain and human placenta using the same methods demonstrating the usefulness of this procedure in obtaining GALC from solid human tissues. In addition to the bands migrating near 50 kDa from urine, there were also bands at 80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of antipeptide antibodies, the 80 kDa band was demonstrated to have the same N-terminal amino acids as the 50-53 kDa bands. The 30 kDa band had a unique sequence. The relationship between the different molecular weight species remains to be determined. The purification of GALC and the securing of amino acid sequence information will aid in the cloning of the GALC gene. This enzyme is deficient in human patients with Krabbe disease and several animal species. 相似文献
20.
2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases. 相似文献