共查询到20条相似文献,搜索用时 31 毫秒
1.
D B Thompson W L Daniel 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,90(4):823-831
1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, Mr = 67 +/- 2 kDa), an apparent aggregate (dog, whale, pig; Mr = 192 +/- 10 kDa), or both (rat, mouse; monomeric Mr = 57 +/- 2 kDa; apparent dimeric Mr = 114 +/- 3 kDa). 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. Opossum arylsulfatases A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations. 相似文献
2.
B Wojczyk 《Biology of the cell / under the auspices of the European Cell Biology Organization》1986,57(2):147-152
Lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolases, EC 3.1.6.1) from horse leukocytes were purified about 680-fold and 70-fold, respectively, starting from a crude extract of the azurophil and specific granules of leukocytes, by affinity, ion exchange, and gel filtration chromatography. Purified arylsulfatase A displayed anomalous kinetics, a pH optimum at 5.2, an isoelectric point at 4.3, and a Km value for p-nitrocatechol sulfate (pNCS) of 0.37 mM. This enzyme was found to exist in two association states depending on pH: a high molecular weight form at pH 5.0 and a low molecular weight form at pH 7.5. Arylsulfatase B displayed normal kinetics, a pH optimum at 5.8, two isoelectric points at pH 8.6 and 8.9, and a Km value for pNCS of 3.38 mM. The thermostability of the two enzymes was different: arylsulfatase B was found to be more stable than arylsulfatase A. Arylsulfatase A was inhibited by sulfate, sulfite, silver, magnesium, manganese and calcium ions and arylsulfatase B by chloride, sulfate, sulfite and silver ions. 相似文献
3.
Homogeneous arylsulfatase A from human placenta, liver and urine contains two nonidentical subunits of 59 and 54 kDa. The two subunits are immunologically identical. The relative amount of low molecular weight subunits is only 20-30% of the total enzyme protein. Treatment of the enzyme under various conditions with endo-beta-N-acetylglucosaminidase F results in a decrease in the apparent molecular weight of both subunits by 1-2 kDa. a value that corresponds to the loss of a single N-linked oligosaccharide. However, as judged by carbohydrate staining, endo-beta-N-acetylglucosaminidase F does not remove all carbohydrate from the subunits or from glycopeptides of arylsulfatase A. In contrast, human prostatic acid phosphatase, a glycoprotein with a high content of mannose, hybrid and complex oligosaccharides is completely deglycosylated under identical experimental conditions. Several attempts to deglycosylate arylsulfatase A by chemical methods were unsuccessful due to poor recovery of the protein. From the present studies we conclude that arylsulfatase A contains an endo-beta-N-acetylglucosaminidase F resistant, perhaps O-linked carbohydrate. 相似文献
4.
A Waheed J M Risley R L Van Etten 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,82(4):855-862
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
5.
N N Ulusu M S Kus N L Acan E F Tezcan 《The international journal of biochemistry & cell biology》1999,31(7):787-796
This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources. 相似文献
6.
Malgorzata Matusiewicz Malgorzata Krzystek-Korpacka 《Journal of experimental marine biology and ecology》2005,317(2):175-187
Arylsulfatase from Artemia salina exists in at least two forms (AS I and AS II). The paper presents characterization of the AS II form of the arylsulfatase. The enzyme was able to hydrolyze p-nitrocatechol sulfate (pNCS) as well as ascorbate sulfate. It exhibited maximum activity at temperature of 50 °C and was stable for 2 h at 4-10 °C. Optimum pH shifted from 6.2 at 4 mM pNCS (substrate) to 4.8 at 20 mM pNCS. The enzyme displayed linear kinetics. AS II arylsulfatase exists in two molecular forms (349 and 460 kDa) composed of identical subunits with molecular mass of 53 kDa. Sulfite and phosphate ions were the most potent inhibitors of the enzyme. Cyanide proved to be a weak inhibitor. Sulfate and low concentrations of silver ions had no effect on the enzyme activity. Based on the above results, modifications in the assay for determination of enzyme activity are proposed. 相似文献
7.
Polygalacturonase isoenzyme 3 (PG-3) was purified to homogeneity with a specific activity of 0.7 mu katal mg-1 protein from banana fruit pulp. The purified enzyme was a glycoprotein with ca. 8% carbohydrate. The molecular weight of the native enzyme was found to be 90 +/- 10 kDa with a subunit molecular weight of 29 +/- 2 kDa. The enzyme exhibited optimum activity at pH 4.3 and temperature 40 degrees C with activation energy 35.4 kJ mol-1. A unique property of the enzyme was the requirement of -SH groups for the enzyme activity. The enzyme was inhibited by p-CMB and activated by 2-ME and DTT. The inhibition of p-CMB could be reversed by DTT. The enzyme contained eight free -SH groups. The Km of the enzyme was 0.15% for polygalacturonic acid. 相似文献
8.
Nergard CS Kiyohara H Reynolds JC Thomas-Oates JE Matsumoto T Yamada H Patel T Petersen D Michaelsen TE Diallo D Paulsen BS 《Biomacromolecules》2006,7(1):71-79
Structures of three pectic arabinogalactans, one from Vernonia kotschyana (Vk2a) and two from Cochlospermum tinctorium (Ct50A1 and Ct50A2), and their complement fixation and induction of B cell proliferation in vitro were compared. The polysaccharide Vk2a expressed potent biological activity in both assays compared with Ct50A1 and Ct50A2. Vk2a possessed a very high molecular weight (1150 +/- 20 kDa) compared with Ct50A1 and Ct50A2 which both showed a polydisperse nature with the highest molecular weight polymers in each fraction estimated at approximately 105 kDa (Ct1a) and 640 +/- 100 kDa (Ct2a), respectively. The HMW polymers showed complement fixation in the same range as the native fractions. The arabinogalactan II content was low in Vk2a (2%) compared with that in Ct50A1 (23%) and Ct50A2 (12%). The high molecular weight polymers were subjected to digestion with a beta-d-(1, 3)-galactanase-rich fraction from Driselase, oligomers were isolated by HPAEC, and their finer structures were determined by MALDI- and ES-qoToF-MS, linkage, and monosaccharide composition analyses. Vk2a consists of both a galacturonan core and a rhamnogalacturonan core rich in neutral side chains. The backbones of both Ct-polysaccharides consist mainly of RG-I regions with numerous neutral side chains dominated by galactosyl residues, whereas the homogalacturonan regions seem to be small. Differences in the chain lengths of the 6-linked galacto-oligosaccharides attached to the 3-linked galactan core could not be related to the differences in the potencies of the biological activities observed. 相似文献
9.
William L. Daniel Berthie M. Ruoff D. Bruce Thompson Julius B. Gore 《Biochemical genetics》1985,23(9-10):771-786
Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable. 相似文献
10.
Brizio C Galluccio M Wait R Torchetti EM Bafunno V Accardi R Gianazza E Indiveri C Barile M 《Biochemical and biophysical research communications》2006,344(3):1008-1016
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast. 相似文献
11.
G Hess 《European journal of biochemistry》1983,135(3):505-509
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
12.
Suárez ER Syvitski R Kralovec JA Noseda MD Barrow CJ Ewart HS Lumsden MD Grindley TB 《Biomacromolecules》2006,7(8):2368-2376
Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity. 相似文献
13.
D B Thompson W L Daniel J H Glaser 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,82(1):55-61
Approximately 25 and 40%, respectively, of murine (Mus musculus) and rat (Rattus norvegicus) hepatic arylsulfatase (EC 3.1.6.1) activity eluted from DEAE-ion exchange resins under high salt conditions. This high salt fraction contained arylsulfatase A and an enzyme which was immunologically similar to arylsulfatase B. The latter enzyme was thermostable, resistant to inhibition by silver, completely inhibited by phosphate, displayed linear kinetics, and had a higher pH optimum than arylsulfatase A. Anionic arylsulfatase B also hydrolyzed chondroitin-4-SO4 heptasaccharide. Sephacryl S-300 gel filtration resolved anionic arylsulfatase B into 55 and 115 kd fractions. Rodent arylsulfatase A activity was grossly underestimated when 4-methyl-umbelliferyl sulfate was employed as substrate. 相似文献
14.
Synthesis and processing of arylsulfatase A in human skin fibroblasts 总被引:10,自引:0,他引:10
A Waheed A Hasilik K von Figura 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1982,363(4):425-430
Biosynthesis of arylsulfatase A in normal and mutant human fibroblasts was studied by growing cells in the presence of L-[4,5-3H] leucine or [2-3H] mannose, isolation of labelled arylsulfatase A by immune precipitation and visualization of electrophoretically separated polypeptide by fluorography. Arylsulfatase A was synthesized as a precursor with a mean apparent molecular mass of 62 kDa. Intracellularly the precursor was converted into a 60.5 kDa polypeptide within a chase period of 1 to 7 days. The 60.5 kDa product in polyacrylamide corresponded to one of two polypeptides present in arylsulfatase A isolated from human placenta. In fibroblasts from a patient with metachromatic leukodystrophy no immune precipitable polypeptides of arylsulfatase A were detected. In normal fibroblasts less than 10% of the precursor of arylsulfatase A was secreted into the medium, whereas in mucolipidosis II fibroblasts and in control fibroblasts grown in the presence of NH4Cl up to 90% of the precursor of arylsulfatase A, appeared in the medium and remained there without change in the apparent molecular mass for at least 7 days. Arylsulfatase A polypeptides appear to contain two carbohydrate side chains. In about 90% of the polypeptides both side chains are cleaved by endo-beta-N-acetylglucosaminidase H, whereas in the remaining chains one of the two oligosaccharides is not cleaved. 相似文献
15.
A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts 总被引:1,自引:0,他引:1
A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface. 相似文献
16.
Characterization of a cyanobacterial photosystem I complex 总被引:3,自引:0,他引:3
A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described. 相似文献
17.
Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities 总被引:1,自引:0,他引:1
The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau 总被引:2,自引:0,他引:2
Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised. 相似文献
19.
T Kobayashi K Honke T Jin S Gasa T Miyazaki A Makita 《Biochimica et biophysica acta》1992,1159(3):243-247
Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation. 相似文献
20.
In addition to their general function in cellular homeostasis, thyroid lysosomes play an essential role in the biosynthesis of thyroid hormones by cleaving the macromolecular prohormone, thyroglobulin. In the present work, we have attempted to determine whether the enzyme composition of thyroid lysosomes differs from that of lysosomes from other tissues. Lysosomal enzymes, cathepsin D, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase, and arylsulfatase A and B, were assayed in crude fractions from various pig tissues, heart, brain, liver, kidney, thyroid, adrenals, ovary, and spleen. It appeared that the specific activity of arylsulfatase A was at least 20 times higher in the thyroid than in most other tissues. Thyroid lysosomes purified by isopycnic centrifugation on Percoll gradients contained two major polypeptides with apparent molecular weights of 58,000 and 54,000 representing about 30% of the total protein. These polypeptides were glycosylated and were exclusively found in the intralysosomal soluble fraction obtained by osmotic pressure-dependent lysis. By fractionating intralysosomal soluble proteins by velocity sedimentation on sucrose gradients or gel permeation chromatography we identified a thyroid arylsulfatase A holoenzyme which corresponds to a 120,000 Mr species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of the gradient or column fractions showed that the 120-kDa protein peak with arylsulfatase A activity essentially contained the 58- and 54-kDa polypeptides in equivalent amounts. In conclusion, arylsulfatase A, a heterodimer of 120 kDa composed of two nonidentical subunits, is the major protein component of thyroid lysosomes. The superabundance of this protein in purified thyroid lysosomes is related to the very high specific activity of the enzyme in the thyroid as compared to other tissues. 相似文献