Mammalian arylsulfatases A and B: relative rates of hydrolysis of artificial substrates

Rodent and bovine arylsulfatase B hydrolyze 4-methylumbelliferyl sulfate (4MUS) 10- to 30-fold more efficiently than arylsulfatase A. Therefore, 4MUS grossly underestimates arylsulfatase A activity in the presence of excess arylsulfatase B.     

Arylsulfatase B synthesis and clearance in inbred mouse strains

Arylsulfatase B activity levels were approximately 2-3-fold higher in adult C57BL/6J liver and kidney compared to corresponding tissues from A/J inbred mice. In vivo incorporation of tritiated leucine into C57BL/6J hepatic arylsulfatase B reached a maximum approximately 15 h after injection. The label was cleared from C57BL/6J arylsulfatase B with an apparent half-life of 36 h. The relative rates of synthesis of C57BL/6J and A/J arylsulfatase B were similar; however, the A/J enzyme was cleared more rapidly from liver tissue. C57BL/6J kidney arylsulfatase B appeared to be synthesized at a 2-3-fold higher rate than the corresponding A/J enzyme. These trends suggest genetic regulation of arylsulfatase B is effected through different means in liver and kidney from adult mice of these two inbred strains.     

Comparative studies of murine anionic and cationic arylsulfatase B (1)

Murine brain possesses an anionic form of arylsulfatase B which accounts for approximately 12-16% of non-microsomal arylsulfatase activity. This isozyme is antigenically similar to cationic arylsulfatase B, displays a similar developmental profile, and can be converted to a form resembling the cationic species by prior treatment with neuraminidase.     

Comparative studies of murine anionic and cationic arylsulfatase B

Summary Murine brain possesses an anionic form of arylsulfatase B which accounts for approximately 12–16% of nonmicrosomal arylsulfatase activity. This isozyme is antigenically similar to cationic arylsulfatase B, displays a similar developmental profile, and can be converted to a form resembling the cationic species by prior treatment with neuraminidase.This research was supported in part by National Institutes of Health Grant: GM 27707.     

Interstrain variation of murine arylsulfatase C.

A method has been developed for the assay of arylsulfatase C in tissue extracts containing arylsulfatases A and B. Significant variation of enzyme activity was observed among 26 inbred murine strains. Activity differences were apparent at all stages evaluated between 1 and 70 days postnatal age. Arylsulfatase C from representative high- and low-activity strains exhibited similar Michaelis constants, temperature optima, pH optima, thermostabilities and inhibitor profiles.     

Murine arylsulfatase C: Evidence for two isozymes

Summary SWR/J mice posses high arylsulfatase C, estrone sulfatase, and dehydroepiandrosterone sulfatase activities in liver, spleen and kidney compared to A/J mice. This internstrain activity variation appears to be determined by at least 1 autosomal gene. Murine arylsulfatase C activity occurs in both hydrophobic and hydrophilic forms which differ with respect to certain biochemical properties and exhibit different subcellular distributions. The hydrophilic isozyme is a major component in kidney and brain extracts and a minor isozyme in liver and spleen extracts. The hydrophobic arylsulfatase C isozyme appears to be identical to steroid sulfatase. The hydrophilic arylsulfatase C isozyme does not possess steroid sulfatase activity; however, hydrophilic and hydrophobic arylsulfatase C share certain properties, suggesting that they may be structurally related. The autosomal gene(s) affects both arylsulfatase isozymes.This research was supported in part by National Institutes of Health grant GM 27707.     

Interstrain variation of murine arylsulfatase C

Summary A method has been developed for the assay of arylsulfatase C in tissue extracts containing arylsulfatases A and B. Significant variation of enzyme activity was observed among 26 inbred murine strains. Activity differences were apparent at all stages evaluated between 1 and 70 days postnatal age. Arylsulfatase C from representative high- and low-activity strains exhibited similar Michaelis constants, temperature optima, pH optima, thermostabilities and inhibitor profiles.This work was supported in part by Biomedical Sciences Support Grant: HEW PHS RR 07030.     

Arylsulfatase in coelomocytes of Holothuria polii

Holothuria polii coelomocytes possess arylsulfatase enzymes. Two pH optima were found for arylsulfatase activity in cell lysate preparations, one at pH 5.0 and the other at pH 5.8. Both increased after injection of zymosan particles or formalinized sheep red blood cells (fSR-BC), indicating an active role of the enzymes during phagocytosis of particulate substances. Under a light microscope, the acid hydrolase arylsulfatase were localized in the granules of spherula cells, and therefore considered lysosomal in nature.     

Differential medium for the isolation and identification ofMycobacterium fortuitum complex

Summary A single medium efficiently selects for an identifiesMycobacterium fortuitum complex on the basis of bile tolerance and arylsulfatase activity.     

Specific determination of arylsulfatase A activity

Summary Arylsulfatase activities in biological materials are too low to be detected by the methods available hitherto. A sensitive and specific assay method for arylsulfatase A (AS-A) has been developed in the present study. Ascorbate-2-sulfate is known to be a specific natural substrate of AS-A; the ascorbic acid liberated by the action of AS-A was quantitatively determined using HPLC equipped with an amperometric detector. The method was used to analyze the activity of AS-A in biological materials.     

Specific determination of arylsulfatase A activity

Arylsulfatase activities in biological materials are too low to be detected by the methods available hitherto. A sensitive and specific assay method for arylsulfatase A (AS-A) has been developed in the present study. Ascorbate-2-sulfate is known to be a specific natural substrate of AS-A; the ascorbic acid liberated by the action of AS-A was quantitatively determined using HPLC equipped with an amperometric detector. The method was used to analyze the activity of AS-A in biological materials.     

Human sulfatases: A structural perspective to catalysis

The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.     

Role of the A chain in thrombin function

The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located > 20 A away from residues of the catalytic triad, the primary specificity pocket and the Na+ site. The R4A mutation causes significant perturbation of Na+ binding, fibrinogen clotting and PAR1 cleavage, but modest reduction of protein C activation in the presence of thrombomodulin. These findings challenge our current paradigm of thrombin structure-function relations focused exclusively on the properties of the catalytic B chain, and explain why certain naturally occurring mutations of the A chain cause serious bleeding.     

Chemical investigation of the seeds ofAlbizzia procera benth

Zusammenfassung Aus dem Samen vonAlbizzia procera Benth wurden ausser Machaerinsäure 4 triterpenoide Sapogenine isoliert (Verbindung A und B, Proceragenin A und B). Es wurde bewiesen, dass die Verbindung A das 28 21-Lacton der Machaerinsäure mit einer Konformationsänderung am Ring D oder E ist, während die Verbindung B als Äthylmachaerinat identifiziert wurde. Bei Proceragenin A und B handelt es sich um Dihydroylactone.

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A part of the work was carried out at the Department of Chemistry, Bose Institute, Calcutta-9, India, and was supported in part by the Ministry of Education, Government of India. — The authors desire to express their sincere thanks to Dr.A. K. Barua, Reader in Chemistry, Bose Institute, Calcutta and to the Rev. Dr.H. Santapau, Director, Botanical Survey of India, Calcutta for their keen interest and encouragement.     

Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B3 to bovine serum albumin. Aflatoxin B3 was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B1 (AFB1) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B1, B2, G1, and G2 when tritiated AFB1 was used as the marker ligand. The concentrations causing 50% inhibition of binding of 3H-AFB1 to the antibodies by unlabeled aflatoxins B1, B2, G1, G2 and B3 were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B1 and G1, two of the most important toxic metabolites produced by Aspergillus flavus and A. parasiticus.     

热压烧结ZrB2／B4C陶瓷材料的组织与性能

以B4C、C、ZrO2为主要原料，采用反应热压法制备ZrB2／B4C陶瓷材料。通过对ZrB2／B4C陶瓷材料的显微组织及力学性能的综合分析，发现第二相ZrB2含量为20wt％时，材料具有较好的综合力学性能，相对密度为99．3％，维氏硬度为36．1GPa，抗弯强度为533．3MPa，断裂韧性为6．95MPa·m^1/2，比纯B4C陶瓷材料的性能均有所提高。材料为穿晶和沿晶断裂的混合断裂模式。     

Methoxyphenylethylamines as substrates for type A and type B monoamine oxidase

Summary 4-Methoxyphenylethylamine was found to be a specific substrate for type B monoamine oxidase (MAO) in rat brain mitochondria, whereas 3,4-dimethoxyphenylethylamine was common for both types of MAO. These results suggest that O-methylation in the para-position increases the preference of the substrate for type B MAO, while a methoxy-group in the meta-position contributes to the substance being a type A substrate.     

A pyruvate kinase variant in different mouse transplanted tumors

Mouse transplanted tumors, in contrast to normal tissues, contain a pyruvate kinase (PK) variant sensitive to the inhibitory action of L-cysteine and less sensitive to saturated fatty acids than the normal enzyme. In selected normal and tumor materials two fractions of PK were separated. Fraction A (20-30% (NH4)2SO4 saturation) dominated in normal liver, and fraction B (50-60% (NH4)2SO4 saturation) in skeletal muscles and Ehrlich ascites tumor. Only this fraction B from tumor material was sensitive to L-cysteine, and seems to contain a tumor-specific PK variant which might be considered as a marker of neoplastic transformation in a broad spectrum of mouse experimental tumors.