Arylsulfatase Activity in Salt Marsh Soils

The presence of arylsulfatase(s) was confirmed in salt marsh soils. The temperatures of maximum activity and inactivation, the pH range over which the enzyme was active, and the K_m values were similar to those of soil enzymes. Unlike soil arylsulfatases, however, the salt marsh enzymes do not appear to be repressed by sulfate. It is postulated that these enzymes may be necessary for the initiation of arylsulfate ester metabolism.     

Effects of ionization of p-nitrocatechol sulfate on its behavior as a substrate for arylsulfatases

Evidence is presented showing that arylsulfatases reacting at acidic pH are more active towards p-nitrocatechol sulfate substrate, while the reverse is true for the enzymes having alkaline pH_max. An explanation of this phenomenon is suggested based on the ionization of p-nitrocatechol sulfate (pK_a = 6.4) and a much higher reactivity of sulfatases with the acidic form of the substrate.     

Screening and Production of Arylsulfatases for Target Therapy with Etoposide 4′-Sulfate,an Antitumor Prodrug

Two arylsulfatase-producing streptomycetes that desulfated etoposide 4′-sulfate were isolated from soil samples. Taxonomical study identified one soil isolate as Streptomyces griseorubiginosus S980-14 (Es-1 arylsulfatase producer), while the other was considered new and tentatively designated Streptomyces sp. T109-3 (Es-2 arylsulfatase producer). Both strains produced extracellular arylsulfatase activities, provided that cultivation media were prepared with distilled water. Unlike the two known types of arylsulfatases, which had significant activity on p-nitrophenyl sulfate but none on etoposide 4′-sulfate, the crude streptomycete arylsulfatases efficiently desulfated etoposide 4′-sulfate and p-nitrophenyl sulfate, which supports the establishment of a new type of arylsulfatases.     

Age determination and estimation of larval period in field caught abalone (Haliotis discus hannai Ino 1953) larvae and newly metamorphosed post-larvae by counts of radular teeth rows

We developed an age determination method for larval and newly metamorphosed post-larval abalone Haliotis discus hannai in a laboratory experiment and determined the age of field caught individuals. Laboratory experiments showed that competent veliger larvae (4 days after fertilization) had a radula and regularly added rows of radular teeth with age in the absence of metamorphosis. Under environmentally relevant temperatures (17-22 °C), the number of rows of radular teeth increased linearly with age, but slopes of the regression lines were different among temperatures. Rows of radular teeth were added more slowly at lower temperatures. The effect of temperature on the development rate of the radula was quantified by the regression and the temperature coefficient, Q₁₀. The radular development of newly metamorphosed post-larvae, which had not acquired a peristomal shell (adult shell), was comparable with that of veliger larvae, although older post-larvae had a larger number of rows of radula than those of the same age of veliger larvae. From these results, an age determination method of veliger larvae and newly metamorphosed post-larvae was established, using the number of rows of radular teeth. The age of veliger larvae and newly metamorphosed post-larvae was determined by the age determination method for samples collected in August to October of 2003 and 2004 for which the thermal history of the coastal water of Miyagi Prefecture Japan was available. Only 9.1% of veliger larvae (n = 8) captured in the field had formed a radula and these were estimated to be 4-6 days old. The remaining 90.9% of larvae (n = 80) that had not formed a radula were classified as younger than 4 days old. All newly metamorphosed post-larvae (n = 24) that had metamorphosed on substrata were estimated to be 4-6 days old. Results of the field study indicate that these abalone metamorphosed within a few days after the acquisition of competence (4 days after fertilization) at this site, which has suitable crustose algal habitat.     

Immunological Studies on Antisera of Mice Immunized with Dried or DEPC-treated Ehrlich Ascites Tumor Cells

Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH₄)₂SO₄, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (V_max) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10^?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.     

Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp.

Organisms able to utilize one of several alkyl- and arylsulfonates as sole source of sulfur under anoxic conditions were enriched. Three fermenting bacteria, all putative Clostridium spp., were isolated in pure culture. All three organisms had wide substrate ranges for alkylsulfonates, taurine and arylsulfonates, presumably due to three different enzyme systems. One organism, strain KNNDS (DSM 10612) was selected for further characterization. The organism was possibly a new Clostridium sp., with Clostidium intestinalis as its nearest neighbor (97.6% similarity of rDNA). Strain KNNDS catalyzed complete sulfonate utilization concomitant with growth. Growth yields of approximtely 3 kg protein/mol sulfur were observed, independent of the sulfur source [e.g. sulfate, sulfide, 4-(phenyl)butyl-1-sulfonate, 2,6-naphthyldisulfonate or 4-nitrocatechol sulfate]. We failed to detect significant amounts of either an arylsulfonatase or an arylsulfatase, and we hypothesize different arylsulfatases [EC 3.1.6.1] in aerobes and in Clostridium spp. Received: 15 October / Accepted: 29 November 1996     

Formation and Purification of Serratia marcescens Arylsulfatase

The effects of culture conditions on arylsulfatase production by six strains of the genus Serratia were studied. Synthesis of arylsulfatases in all six strains was repressed in media with inorganic sulfate or methionine as the sole source of sulfur and derepressed by the addition of tyramine. Serratia marcescens IFO 3046 grew most rapidly and produced a high level of arylsulfatase when cultured on mannitol with inorganic sulfate and tyramine. The derepressed synthesis of arylsulfatase in S. marcescens was not subject to strong catabolite repression. The molecular weight of purified arylsulfatase was determined to be between 46,000 and 49,000. Arylsulfatase from S. marcescens differed in K_m and V_max values, substrate specificities, fluoride inhibition, and electrophoretic mobility from the enzyme from K. aerogenes, but had the same molecular weight as the latter.     

Ontogeny of gut morphology in the white shrimp Penaeus setiferus (Decapoda,Penaeidae)

Ontogeny of the gut in Penaeus setiferus was investigated by reconstruction of serial sections examined by light microscopy. Development of the gut into the adult form is protracted over several weeks beyond metamorphosis in steps that may be directly related to the unique postlarval life history of Penaeus. The gastric mill is lacking in larval stages of P. setiferus. In protozoeal stages Z₁-Z₃, the pyloric ampullae are blind sacs that do not communicate with the midgut. The gland filter first appears in mysis stage M₂. The gastric mill in early postlarval (PL) stages consists of poorly chitinized lobes with flexible setae. By PL₂₁ the ossicles of the gastric mill are rigid and setae are replaced by spine-like denticles, but even by PL₃₅ the gastric mill is neither as massive nor heavily chitinized as in adults. During the mysis stages and early PL stages, the hepatopancreas communicates freely with both the foregut and the midgut trunk. By PL₃₅ the hepatopancreatic ducts are essentially isolated from the remainder of the midgut by foregut ossicles. The midgut in Z₁ consists of two pairs of simple caeca and the midgut trunk. During larval growth, each of the lateral midgut caeca develops into a number of lobes. After metamorphosis these lobes begin to ramify into small-diameter tubules, and by PL₃₅ have completely ramified into the hepatopancreas of adults. From M₁ to PL₄, the anterior midgut caeca decrease in absolute size and become a single anterior diverticulum. The posterior midgut diverticulum first appears in PL₂₁ as a simple sac and thereafter increases in size and complexity.     

Compartmentalization and regulation of arylsulfatase activities in Streptomyces sp., Microbacterium sp. and Rhodococcus sp. soil isolates in response to inorganic sulfate limitation

Arylsulfatases allow microorganisms to satisfy their sulfur (S) requirements as inorganic sulfate after sulfate ester hydrolysis. Our objectives were to investigate the arylsulfatase activities among soil isolates, especially Streptomyces sp., Microbacterium sp. and Rhodococcus sp., because such investigations are limited for these bacteria, which often live in sulfate-limited conditions. Physiological and biochemical analyses indicated that these isolates possessed strong specific arylsulfatase activities ranging from 6 to 8 U. Moreover, for Streptomyces sp., an arylsulfatase localization study revealed 2 forms of arylsulfatases. A first form was located in the membrane, and a second form was located in the intracellular compartment. Both arylsulfatases had different patterns of induction. Indeed, the intracellular arylsulfatase was strictly induced by inorganic sulfate limitation, whereas the membrane arylsulfatase was induced both by substrate presence or S demand independently. For Microbacterium and Rhodococcus isolates, only a membrane arylsulfatase was found. Consequently, our results suggest the presence of a previously undescribed arylsulfatase in these microorganisms that allows them to develop an alternative strategy to fulfill their S requirements compared to bacteria previously studied in the literature.     

Comparison of growth and lipid composition in the green abalone, Haliotis fulgens, provided specific macroalgal diets

Lipid composition of abalone was examined over a one-year interval. A feeding trial was designed to cover a full reproductive cycle in young adult green abalone, Haliotis fulgens, consisting of five diet treatments: the macrophytic algal phaeophyte Egregia menziesii, rhodophyte Chondracanthus canaliculatus, chlorophyte Ulva lobata, a composite of the three algae and a starvation control. The lipid class, fatty acid, sterol and 1-O-alkyl glyceryl ether profiles were determined for foot, hepatopancreas/gonad tissues and larvae. The major fatty acids were 16:0, 18:0, 18:1(n-7)c, 18:1(n-9)c, 20:4(n-6), 20:5(n-3) and 22:5(n-3), as well as 14:0 for abalone fed brown and red algae. 4,8,12-Trimethyltridecanoic acid, derived from algae, was detected for the first time in H. fulgens (hepatopancreas complex, 1.2–13.9%; larvae, 0.5% of total fatty acids). Diacylglyceryl ethers were present in larvae (0.6% of total lipid). The major 1-O-alkyl glycerols were 16:0, 16:1 and 18:0. Additionally, 18:1(n-9) was a major component in hepatopancreas/gonad and larvae. The major sterol was cholesterol (96–100% of total sterols). Highest growth rates were linked to temperature and occurred in abalone fed the phaeophyte E. menziesii (43 μm·day^–1, 56 mg·day^–1 yearly mean), an alga containing the highest levels of C₂₀ polyunsaturated fatty acids and the highest ratio of 20:4(n-6) to 20:5(n-3). This study provides evidence of the influence of diet and temperature on seasonal changes in abalone lipid profiles, where diet is most strongly related to body mass and temperature to shell length. The allocation of lipids to specific tissues in green abalone clarifies their lipid metabolism. These results provide a basis for improving nutrition of abalone in mariculture through formulation of artificial feeds.     

Recognition of arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-phosphotransferase

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.     

Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency

Multiple-sulfatase deficiency (MSD) is now considered to be heterogeneous and could be classified into three or four clinical phenotypes according to the onset of the disease: neonatal, late infantile, juvenile and possibly adult type. Neonatal-type MSD shows severe clinical involvement and practically no arylsulfatase A, B and C activities in cultured skin fibroblasts. Furthermore, arylsulfatase A activity in neonatal-type MSD was not enhanced by the addition of thiosulfate. Therefore, it is distinct from late infantile-type MSD. The degradation of 14C-sulfatide can occur in MSD-cultured skin fibroblasts and was much higher than in late infantile-type MLD. The addition of thiol protease such as leupeptin to cultured MSD skin fibroblasts enhanced arylsulfatase A activity as well as the degradation of 14C-sulfatide. This suggests that the decreased activities of arylsulfatase A is due to an acceleration of the enzyme degradation. Enzyme replacement by the addition of arylsulfatases of different sources (human liver, brain, fungus) was carried out in cultured MSD skin fibroblasts. Human enzymes of arylsulfatase A and B were mostly taken up by MSD cells rather than those of fungus origin. By the exposure to leukocytes to cultured skin fibroblasts, MSD cells corrected arylsulfatase A and B activities.     

Transformations histologiques lors de la métamorphose chez Cymbulia peroni de Blainville (Mollusca,Opisthobranchia)

The histological structure of the veliger of Cymbulia peroni is described at the stage close to metamorphosis and during the metamorphosis. Major transformations are observed in the tegument and in the digestive system.     

Biochemical characterization of neonatal multiple sulfatase deficient (MSD) disorder cultured skin fibroblasts

Cultured skin fibroblasts of a patient with a neonatal onset of MSD can be distinguished from usual type of MSD cells by enzyme assays of arylsulfatases A, B and C activities, effect of thiosulfate on arylsulfatase A activity and ³⁵SO₄-acid mucopolysaccharide accumulation and degradation. These data suggest that neonatal onset of MSD is a distinct disorder from usual type of MSD in term of genetic occurence.     

Improved high-performance liquid chromatographic method for N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B) activity determination using uridine diphosho-N-acetylgalactosamine-4-sulfate

UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 μmol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation: (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination ?3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K_m=85 μM.     

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.     

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.     

Isolation and characterization of octopus hepatopancreatic glutathione S-transferase. Comparison of digestive gland enzyme with lens S-crystallin

Glutathione S-transferase fromOctopus vulgaris hepatopancreas was purified to apparent homogeneity by single glutathione-Sepharose-4B affinity chromatography with overall yield 46% and purification 249-fold. The enzyme was a homodimer with subunitM _r 24,000, which was smaller than that of the octopus lens S-crystallin (M _r 27,000) with glutathione-S-transferase-like structure. Both proteins showed substrate specificities similar to/-type isozyme of glutathione S-transferase. Under native conditions, both proteins exhibited multiple forms upon polyacrylamide gel electrophoresis or isoelectric focusing, albeit with distinct mobilities; however, only one kind of N-terminal amino acid sequence was determined for the multiple forms of each protein. The hepatopancreatic GST, withpI value 6.6–7.3, dissociated into two monomers in an acidic or alkaline environment. Two amino acid residues, withpK _a values 5.69±0.14 and 9.03±0.11 were involved in the subunit interactions of the hepatopancreatic enzyme.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - IEF isoelectric focusing - GSH glutathione - GST glutathione S-transferase - CDNB 1-chloro-2,4-dinitrobenzene - EA ethacrynic acid [2,3-dichloro-4-(2-methylenebutyryl) phenoxy)acetic acid]     

Catabolism of 1,3-dinitrobenzene by Rhodococcus sp. QT-1

The 1,3-dinitrobenzene-degrading Rhodococcus strain QT-1 was isolated under nitrogen limiting conditions from contaminated soil samples. Experimental data indicate that 1,3-dinitrobenzene is metabolized via 4-nitrocatechol. Both compounds were oxidized by resting cells and nitro groups were completely eliminated as nitrite. Strain QT-1 utilizes both 1,3-dinitrobenzene and 4-nitrocatechol as source of nitrogen in the absence as well as in the presence of high amounts of ammonia. Growth on 4-nitrocatechol does not induce the enzyme(s) for the initial oxidation of 1,3-dinitrobenzene.Abbreviations TNT 2,4,6-trinitrotoluene - 1,3DNB 1,3-dinitrobenzene - 4NC 4-nitrocatechol - 3NA 3-nitroaniline - NB nutrient broth; t_d doubling time - OD₅₄₆ optical density at 546 nm