首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.  相似文献   

2.
The biosynthesis of arylsulfatase A in human skin fibroblasts was studied by labeling cells and isolating arylsulfatase A using immune precipitation and polyacrylamide gel electrophoresis under denaturing and reducing conditions. Arylsulfatase A was synthesized as precursor polypeptides of 62 kDa or 59.5 kDa. Cell lines synthesizing either or both polypeptides were found. The results of a family study were consistent with the assumption that the two arylsulfatase A polypeptides are of allelic nature. In various heterozygous cell lines, the two polypeptides were formed at equal or different rates. The relative rate of biosynthesis was constant for an individual cell line, suggesting that both allelic products were under separate genetic control. In a group of 21 unrelated individuals, the gene frequency of alleles for the 62- and 59.5-kDa precursor forms was 3:1. The two allelic forms of the arylsulfatase A polypeptides were converted into a 57-kDa form by endo-beta-N-acetylglucosaminidase H, an enzyme specifically removing asparagine-linked oligosaccharides of the high-mannose (and hybrid) type. The apparent difference in the number of asparagine-linked oligosaccharides suggests that the two allelic genes differ in a region coding the sequence Asn-X-Thr(Ser), which is required for attachment of asparagine-linked oligosaccharides.  相似文献   

3.
Using a combination of differential centrifugation and isopycnic centrifugation in Percoll gradients, we obtained a highly purified preparation of thyroid lysosomes [Alquier, Guenin, Munari-Silem, Audebet & Rousset (1985) Biochem. J. 232, 529-537] in which we identified thyroglobulin. From this observation, we postulated that the isolated lysosome population could be composed of primary lysosomes and of secondary lysosomes resulting from the fusion of lysosomes with thyroglobulin-containing vesicles. In the present study, we have tried to characterize these lysosome populations by (a) subfractionation of purified lysosomes using iterative centrifugation on Percoll gradients and (b) by functional studies on cultured thyroid cells. Thyroglobulin analysed by soluble phase radioimmunoassay, Western blotting or immunoprecipitation was used as a marker of secondary lysosomes. The total lysosome population separated from other cell organelles on a first gradient was centrifuged on a second Percoll gradient. Resedimented lysosomes were recovered as a slightly asymmetrical peak under which the distribution patterns of acid hydrolase activities and immunoreactive thyroglobulin did not superimpose. This lysosomal material (L) was separated into two fractions: a light (thyroglobulin-enriched) fraction (L2) and a dense fraction (L1). L1 and L2 subfractions centrifuged on a third series of Percoll gradients were recovered as symmetrical peaks at buoyant densities of 1.12-1.13 and 1.08 g/ml, respectively. In each case, protein and acid hydrolase activities were superimposable. The specific activity of acid phosphatase was slightly lower in L2 than in L1. In contrast, the immunoassayable thyroglobulin content of L2 was about 4-fold higher than that of L1. The overall polypeptide composition of L, L1 and L2 analysed by polyacrylamide-gel electrophoresis was very similar, except for thyroglobulin which was more abundant in L2 than in either L or L1. The functional relationship between L1 and L2 lysosome subpopulations has been studied in cultured thyroid cells reassociated into follicles. Thyroid cells, prelabelled with 125I-iodide to generate 125I-thyroglobulin, were incubated in the absence of in the presence of inhibitors of intralysosomal proteolysis. The fate of 125I-thyroglobulin, and especially its appearance in the lysosomal compartment, was studied by Percoll gradient fractionation and immunoprecipitation. Treatment of prelabelled thyroid cells with chloroquine and leupeptin induced the accumulation of immunoprecipitable 125I-thyroglobulin into a lysosome fraction corresponding to the L2 subpopulation. In control cells, in which intralysosomal proteolysis was n  相似文献   

4.
Human liver arylsulfatase A was resolved into six fractions by narrow pH range preparative isoelectric focusing. Analytical isoelectric focusing revealed that most enzyme fractions were composed of two adjacent charge isomers. Nevertheless, there was considerable enrichment of charge species which allowed a comparative study of selected properties. Except for the most cationic fraction, neuraminidase treatment converted enzyme in all fractions to the three most cationic species. The most electronegative enzyme species had the highest molecular mass being made up of 64-kDa subunits. As electronegativity decreased, there was concomitant decrease in molecular mass and increase in complexity of subunit composition. Two subunits--61 and 55 kDa--prevailed with increasing proportions of the smaller unit with loss of electronegativity. There was also an increasing amount of a 26-kDa fraction which became a substantial component of the most cationic subfraction. Only enzyme in the two fractions containing the largest and most anionic species were taken up by cultured fibroblasts at higher efficiency than unfractionated enzyme. It is suggested that processing or maturation of arylsulfatase A incurs stepwise removal of charge groups and/or peptide segments leading to smaller, less-charged enzyme species.  相似文献   

5.
Summary It had been shown previously that arylsulfatase A activity was attenuated in pseudo arylsulfatase A deficiency fibroblasts and that subunits of the enzyme were smaller than subunits of the enzyme in normal fibroblasts. Attenuated enzyme activity has now been affirmed in other tissues. Subunits of the enzyme from these sources were also found to be smaller with apparent molecular size 59 and 56 kdaltons. Subunits of enzyme in corresponding control tissues were larger and there was heterogeneity in apparent molecular size as follows: fibroblast, 63 and 59 kdaltons; liver, 63 and 59 kdaltons; kidney, 62 and 58 kdaltons; and urine, 61 and 57 kdaltons. Attenuated enzyme activity and structurally altered enzyme in pseudo arylsulfatase A deficiency appears to be systemic. However, the reason for reduced amounts of structurally altered enzyme with normal catalytic activity is unresolved.  相似文献   

6.
Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.  相似文献   

7.
Cloning and expression of human arylsulfatase A   总被引:25,自引:0,他引:25  
A full length cDNA for human arylsulfatase A was cloned and sequenced. The predicted amino acid sequence comprises 507 residues. A putative signal peptide of 18 residues is followed by the NH2-terminal sequence of placental arylsulfatase A. One of the arylsulfatase A peptides ends 3 residues ahead of the predicted COOH terminus. This indicates that proteolytic processing of arylsulfatase A is confined to the cleavage of the signal peptide. The predicted sequence contains three potential N-glycosylation sites, two of which are likely to be utilized. The sequence shows no homology to any of the known sequences of lysosomal enzymes but a 35% identity to human steroid sulfatase. Transfection of monkey and baby hamster kidney cells resulted in an up to 200-fold increase of the arylsulfatase A activity. The arylsulfatase A was located in lysosome-like structures and transported to dense lysosomes in a mannose 6-phosphate receptor-dependent manner. The arylsulfatase A cDNA hybridizes to 2.0- and 3.9-kilobase species in RNA from human fibroblasts and human liver. RNA species of similar size were detected in metachromatic leukodystrophy fibroblasts of two patients, in which synthesis of arylsulfatase A polypeptides was either detectable or absent.  相似文献   

8.
Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.  相似文献   

9.
Fibroblasts from I-cell disease, a genetically-determined lysosomal storage disease, are shown to contain large amounts of phase-dense lysosomes. These lysosomes accumulated acridine orange and were specifically labeled with antibodies to arylsulfatase A. In normal skin fibroblasts the number of arylsulfatase-containing lysosomes was considerably lower. By immunocytochemistry, metabolic labeling and enzyme assay, the arylsulfatase A in I-cell fibroblasts was shown to be synthesized, stored and secreted at a level that was several-fold higher than that present in heterozygous I-cell or normal fibroblasts. Arylsulfatase A in I-cell fibroblasts differed from arylsulfatase in normal fibroblasts by the absence of endoglycosidase H-sensitive phosphorylated oligosaccharides. These findings indicate that arylsulfatase A in I-cells is targeted to lysosomes by a mechanism that does not appear to involve the phosphorylated mannose marker.  相似文献   

10.
Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.  相似文献   

11.
Membrane-bound 4-beta-galactosyltransferase (lactose synthase; UDP galactose: D-glucose 4-beta-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70,000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.  相似文献   

12.
A 2.2-kilobase cDNA clone for human arylsulfatase B (ASB) and several genomic clones were isolated and sequenced. The deduced amino acid sequence of 533 amino acids contains a 41-amino acid N-terminal signal peptide and a mature polypeptide of 492 amino acid residues. Overexpression of ASB in transfected baby hamster kidney (BHK) cells resulted in up to 68-fold higher ASB activity than in untransfected BHK cells. Pulse-chase labeling showed that ASB was synthesized and secreted as a 64-kDa precursor and processed to a 47-kDa mature form in BHK cells. The 47-kDa ASB form was located in dense lysosomes. Transport of ASB to the lysosomes was accomplished in a mannose 6-phosphate receptor-dependent manner. The ASB cDNA clone hybridizes to 4.8-, 2.5-, and 1.8-kilobase species of RNA from human fibroblasts. The same pattern was observed in RNA from fibroblasts of three Maroteaux-Lamy patients who were deficient in ASB activity, as well as in RNA from fibroblasts of three patients with multiple sulfatase deficiency, in which all known sulfatases were markedly diminished. Deduced amino acid sequences of human arylsulfatase A, human ASB, human steroid sulfatase, human glucosamine-6-sulfatase, and an arylsulfatase from sea urchin showed a substantial degree of similarity suggesting that they arose from a common ancestral gene and are members of an arylsulfatase gene family.  相似文献   

13.
A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 A, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, calmodulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logarithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50,000 to 60,000 which can bind to calmodulin. Thus a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.  相似文献   

14.
Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue-Sepharose affinity chromatography, DEAE-cellulose chromatography, hydrophobic chromatography on phenyl-Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis. The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction. The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.  相似文献   

15.
Rabbit liver arylsulfatase A (arylsulfatase sulfohydrolase, EC 3.1.6.1) monomer was immobilized on cyanogen bromide-activated Sepharose-6MB and on Affi-Gel-10 under various experimental conditions in order to study the effects of variables in sulfatase monomer/oligomer subunit affinity chromatography. First, the number of reactive groups on activated Sepharose-6MB and Affi-Gel-10 was determined by a procedure involving spectrophotometric titration with L-tyrosine. After covalent coupling of sulfatase monomers to the gels, the enzyme binding capacities of the sulfatase subunit affinity gel matrixes were determined at pH 4.5. The maximum binding of free monomers from solution could be achieved when the Affi-Gel-10 protein monomer matrix was prepared at low degrees of covalent loading. The introduction of a batch technique for equilibration of the protein sample with the monomer affinity matrix also increased the efficiency of the subunit affinity gel in purification procedures. The effect of pH on the stability of the heterodimers formed between monomers of rabbit liver arylsulfatase A immobilized on Affi-Gel-10 and free monomers of arylsulfatase A enzymes from different tissues and organisms was studied using the batch technique. For all sulfatase A enzymes tested, the midpoint of the pH transition for subunit association was pH 6.2, suggesting that the amino acid residues involved in the dimerization are similar. The versatility of the Affi-Gel-10 monomer affinity matrix was further demonstrated by purifying 13 mammalian arylsulfatase A enzymes to homogeneity, as assessed by Sephacryl chromatography, native and SDS gel electrophoresis. The molecular weights of the homogeneous monomers and their peptide subunits were in the range of 110-180 KDa and 50-64 KDa, respectively. The amino acid compositions of these enzymes were also determined.  相似文献   

16.
Spermatozoa of sturgeons (Acipenseriformes), unlike teleosts, possess an acrosome. This paper provides data concerning biochemical characteristics of arylsulfatase (AS), an acrosomal enzyme, found in Russian sturgeon spermatozoa and seminal plasma. The enzymes were purified by a four-step procedure, using n-butanol extraction, ion-exchange chromatography repeated twice and gel filtration. High purity of our enzymes was confirmed by silver staining electrophoresis and an immunological experiment. Kinetic parameters indicated that the purified enzymes belong to arylsulfatase type A. Similarity of the seminal plasma arylsulfatase to the spermatozoan enzyme showed us that arylsulfatase from seminal plasma might originate from damaged spermatozoa. The possible physiological consequences of the presence of arylsulfatase in Russian sturgeon semen are discussed.  相似文献   

17.
Starch branching enzyme (SBE, EC 2.4.1.18) activity was followed in developing barley ( Hordeum vulgare L. cv. Golf) caryopses during a period of one month after anthesis. Caryopses with the highest specific activity, and corresponding to a fresh weight of around 60 mg per caryopsis, were homogenized and the soluble extract used for branching enzyme purification by FPLC chromatography. Four branching enzyme activity fractions were resolved. From one of these fractions, which exhibited high activity in both the phosphorylation stimulation and amylose branching assays, a branching enzyme preparation containing two related polypeptides of 51 and 50 kDa was obtained. Native polyacrylamide gel electrophoresis and gel filtration showed that the 51/50-kDa polypeptide is monomeric. A combination of phosphorylation stimulation and amylose branching gel assays, SDS-PAGE, and TLC was used to demonstrate the branching activity of the 51/50-kDa polypeptide. The activity was further confirmed by spectroscopic analyses of iodine-glucan complexes. SBEs from four different plant species were compared using the phosphorylation stimulation gel assay.  相似文献   

18.
1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.  相似文献   

19.
Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-A resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号