Assuntos
Anticoagulantes/efeitos adversos , Arilsulfatases/efeitos dos fármacos , Condrodisplasia Punctata/induzido quimicamente , Condrodisplasia Punctata/diagnóstico por imagem , Varfarina/efeitos adversos , Arilsulfatases/fisiologia , Fêmur/diagnóstico por imagem , Falanges dos Dedos da Mão/diagnóstico por imagem , Humanos , Vértebras Lombares/diagnóstico por imagem , Ossos Pélvicos/diagnóstico por imagem , RadiografiaRESUMO
The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C(alpha)-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.
Assuntos
Arilsulfatases/fisiologia , Regulação Enzimológica da Expressão Gênica , Lisossomos/enzimologia , Sulfatases/química , Arilsulfatases/química , Linhagem Celular Tumoral , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Rim/enzimologia , Fígado/enzimologia , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Modelos Biológicos , Pâncreas/enzimologia , Ligação Proteica , Sulfatos/químicaRESUMO
The inclusion of a mutation in a pathology-based database such as the Human Gene Mutation Database (HGMD) is a two-stage process: first, the mutation must occur at the DNA level, then it must cause a clinically detectable disease state. The likelihood of the latter step, termed the relative clinical observation likelihood (RCOL), can be regarded as a function of the structural/functional consequences of a mutation at the protein level. Following this paradigm, we modeled in silico all amino acid replacements that could potentially have arisen from an inherited single base pair substitution in five human genes encoding arylsulphatase A (ARSA), antithrombin III (SERPINC1), protein C (PROC), phenylalanine hydroxylase (PAH), and transthyretin (TTR). These proteins were chosen on the basis of 1) the availability of a crystallographic structure, and 2) a sufficiently large number of amino acid replacements being logged in HGMD. A total of 9,795 possible mutant structures were modeled and 20 different biophysical parameters assessed. Together with the HGMD-derived spectra of clinically detected mutations, these data allowed maximum likelihood estimation of RCOL profiles for the 20 parameters studied. Nine parameters (including energy difference between wild-type and mutant structures, accessibility of the mutated residue, and distance from the binding/active site) exhibited statistically significant variability in their RCOL profiles, indicating that mutation-associated changes affected protein function. As yet, however, a biological meaning could only be attributed to the RCOL profiles of solvent accessibility and, for three proteins, local energy change, disturbed geometry, and distance from the active center. The limited ability of the biophysical properties of mutations to explain clinical consequences is probably due to our current lack of understanding as to which amino acid residues are critical for protein folding. However, since the proteins examined here were unrelated, and our findings consistent, it may nevertheless prove possible to extrapolate to other proteins whose dysfunction underlies inherited disease.
Assuntos
Substituição de Aminoácidos/genética , Biofísica/métodos , Doenças Genéticas Inatas/genética , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/metabolismo , Antitrombina III/química , Antitrombina III/genética , Antitrombina III/fisiologia , Deficiência de Antitrombina III/genética , Deficiência de Antitrombina III/metabolismo , Arilsulfatases/química , Arilsulfatases/deficiência , Arilsulfatases/genética , Arilsulfatases/fisiologia , Bases de Dados Genéticas , Bases de Dados de Proteínas , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/metabolismo , Genótipo , Humanos , Leucodistrofia Metacromática/enzimologia , Leucodistrofia Metacromática/genética , Modelos Genéticos , Mutação de Sentido Incorreto/genética , Fenótipo , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/fisiologia , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genética , Pré-Albumina/química , Pré-Albumina/deficiência , Pré-Albumina/genética , Pré-Albumina/fisiologia , Proteína C/química , Proteína C/genética , Proteína C/fisiologia , Deficiência de Proteína C/genética , Deficiência de Proteína C/metabolismo , Estrutura Secundária de Proteína/genéticaRESUMO
Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute to E. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous to E. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1 aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved when aslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.
Assuntos
Arilsulfatases/genética , Encéfalo/microbiologia , Endotélio Vascular/microbiologia , Escherichia coli/genética , Genes Bacterianos , Sequência de Aminoácidos , Arilsulfatases/fisiologia , Sequência de Bases , Encéfalo/irrigação sanguínea , Células Cultivadas , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Endotélio Vascular/citologia , Escherichia coli/patogenicidade , Teste de Complementação Genética , Humanos , Lactente , Dados de Sequência Molecular , VirulênciaRESUMO
Steroid sulfatase (STS) hydrolyzes several sulfated steroids such as estrone sulfate, dehydroepiandrosterone sulfate, and cholesterol sulfate. In the present study, we have measured STS mRNA levels in 97 breast cancers by reverse transcription-PCR using a fluorescent primer in the presence of an internal standard RNA and evaluated its association with disease-free and overall survival. The median value was 728.0 amol/ng RNA (range, 0-11,778 amol/ng RNA). Levels were significantly higher in tumors demonstrating lymph node metastasis than in those without nodal involvement (P = 0.033) and in patients who experienced a recurrence during the follow-up period (mean, 40.8 months; median, 39 months) compared with those with no evidence of further disease (mean, 49.2 months; median, 48 months; P = 0.029). No significant associations were found between STS mRNA expression and age, menopausal status, tumor size, histological grade, estrogen receptor status, or postoperative adjuvant therapy. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival as a continuous variable (log STS mRNA; P = 0.028). As a dichotomous variable with an optimized cutoff point of 1,240 amol/ng RNA, expression was also associated with a significantly shorter relapse-free survival rate (P = 0.002), but no significant correlation was found between the STS mRNA level and overall survival. Expression was found to be an independent factor for predicting relapse-free survival on multivariate analysis. The results thus support a putative role of STS in breast cancer growth and metastasis.
Assuntos
Arilsulfatases/genética , Neoplasias da Mama/enzimologia , Recidiva Local de Neoplasia/enzimologia , Adulto , Idoso , Arilsulfatases/fisiologia , Neoplasias da Mama/mortalidade , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptores de Estrogênio/análise , Esteril-Sulfatase , Taxa de SobrevidaRESUMO
Cholesterol sulfate is a multifunctional sterol metabolite, produced in large amounts in squamous keratinizing epithelia. Because patients with recessive x-linked ichthyosis display not only a 10-fold increase in cholesterol sulfate, but also a 50% reduction in cholesterol, we assessed here whether cholesterol sulfate accumulation and/or cholesterol deficiency produce abnormal barrier function in recessive x-linked ichthyosis. Patients with recessive x-linked ichthyosis display both an abnormal barrier under basal conditions, and a delay in barrier recovery after acute perturbation, which correlate with minor abnormalities in membrane structure and extensive lamellar-phase separation. Moreover, both the functional and the structural abnormalities were corrected by topical cholesterol. Yet, topical cholesterol sulfate produced both a barrier abnormality in intact skin and extracellular abnormalities in isolated stratum corneum, effects largely reversed by coapplications of cholesterol. Together, these results suggest that cholesterol sulfate accumulation rather than cholesterol deficiency is responsible for the barrier abnormality. Despite the apparent importance of cholesterol sulfate-to-cholesterol processing for normal barrier homeostasis, neither steroid sulfatase activity nor mRNA levels are upregulated following acute perturbations. These results demonstrate both a potential role for cholesterol sulfate-to-cholesterol processing in normal permeability barrier homeostasis, and that basal levels of steroid sulfatase are sufficient to accommodate acute insults to the permeability barrier.
Assuntos
Ictiose Ligada ao Cromossomo X/fisiopatologia , Pele/citologia , Administração Tópica , Adolescente , Adulto , Idoso , Animais , Arilsulfatases/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ésteres do Colesterol/metabolismo , Ésteres do Colesterol/farmacologia , Espaço Extracelular/fisiologia , Humanos , Masculino , Camundongos , Camundongos Pelados , Pessoa de Meia-Idade , Esteril-SulfataseRESUMO
Trichloroethylene (TCE) and related hydrocarbons constitute an important class of environmental pollutants whose adverse effects on liver, kidney, and other tissues may, in part, be mediated by peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors belonging to the steroid receptor superfamily. Activation of PPAR induces a dramatic proliferation of peroxisomes in rodent hepatocytes and ultimately leads to hepatocellular carcinoma. To elucidate the role of PPAR in the pathophysiologic effects of TCE and its metabolites, it is important to understand the mechanisms whereby PPAR is activated both by TCE and endogenous peroxisome proliferators. The investigations summarized in this article a) help clarify the mechanism by which TCE and its metabolites induce peroxisome proliferation and b) explore the potential role of the adrenal steroid and anticarcinogen dehydroepiandrosterone 3beta-sulfate (DHEA-S) as an endogenous PPAR activator. Transient transfection studies have demonstrated that the TCE metabolites trichloroacetate and dichloroacetate both activate PPAR alpha, a major liver-expressed receptor isoform. TCE itself was inactive when tested over the same concentration range, suggesting that its acidic metabolites mediate the peroxisome proliferative potential of TCE. Although DHEA-S is an active peroxisome proliferator in vivo, this steroid does not stimulate trans-activation of PPAR alpha or of two other PPAR isoforms, gamma and delta/Nuc1, when evaluated in COS-1 cell transfection studies. To test whether PPAR alpha mediates peroxisomal gene induction by DHEA-S in intact animals, DHEA-S has been administered to mice lacking a functional PPAR alpha gene. DHEA-S was thus shown to markedly increase hepatic expression of two microsomal P4504A proteins associated with the peroxisomal proliferative response in wild-type mice. In contrast, DHEA-S did not induce these hepatic proteins in PPAR alpha-deficient mice. Thus, despite its unresponsiveness to steroidal peroxisome proliferators in transfection assays, PPAR alpha is an obligatory mediator of DHEA-S-stimulated hepatic peroxisomal gene induction. DHEA-S, or one of its metabolites, may thus serve as an important endogenous regulator of liver peroxisomal enzyme expression.
Assuntos
Hidrocarbonetos Clorados/efeitos adversos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Esteroides/fisiologia , Fatores de Transcrição/efeitos dos fármacos , Animais , Arilsulfatases/fisiologia , Indução Enzimática , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Microssomos Hepáticos , Plasmídeos , Coelhos , Esteril-Sulfatase , Transfecção , Tricloroetileno/efeitos adversosRESUMO
The physiological role of arylsulfatase (Ars) and its function during development have yet to be satisfactorily defined in any species, though the proteins are widely distributed and the genes have been cloned from various organisms. Here we report the dual location of two types of Ars in sea urchin embryos. The majority of sea urchin Ars does not exhibit enzyme activity and is extracellularly distributed in aboral ectoderm cells (nonenzymatic Ars). Only a small portion has enzyme activity and is localized in lysosomal vesicles (enzymatic Ars). The elution pattern of Ars proteins processed by DEAE-cellulose or analytical gel-column chromatography reveals that although the molecular radius of enzymatic Ars differs from that of nonenzymatic Ars, they have the same charge. Furthermore, sedimentation analysis shows that purified Ars of sea urchin embryos is soluble in the absence of divalent cations but becomes insoluble in the presence of Ca2+ or Mg2+. Taken together, the present results suggest that non-enzymatic Ars is a new member of the cell surface component or extracellular matrix. It is possible that this cell surface Ars plays an important role in morphogenesis of sea urchin embryos.
Assuntos
Arilsulfatases/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Ouriços-do-Mar/embriologia , Animais , Arilsulfatases/química , Arilsulfatases/fisiologia , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Proteínas de Membrana/química , Ouriços-do-Mar/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologiaRESUMO
X-linked ichthyosis is the result of steroid sulfatase (STS) deficiency. While most affected individuals have extensive deletions of the STS gene, point mutations have been reported in three patients (1). In this study, we identify an additional three point mutations and characterize the effects of all six mutations on STS activity and expression. All six are unique single base pair substitutions. The mutations are located in a 105-amino acid region of the C-terminal half of the polypeptide. Five of the six mutations involve the substitutions of Pro or Arg for Trp372, Arg for His444, Tyr for Cys446, or Leu for Cys341. The other mutation is in a splice junction and results in a frameshift causing premature termination of the polypeptide at residue 427. All the affected residues are conserved to some degree within the sulfatase family. The six mutations were reproduced in normal STS cDNA and transiently expressed in STS-deficient cells. All six mutant vectors direct the expression of STS protein that lacks enzymatic activity. The mutant polypeptides show a shift in mobility on SDS-PAGE and resistance to proteinase K digestion when translated in the presence of dog pancreas microsomes, indicating glycosylation and normal translocation.
Assuntos
Arilsulfatases/genética , Ictiose Ligada ao Cromossomo X/genética , Mutação Puntual , Animais , Arilsulfatases/química , Arilsulfatases/fisiologia , Linhagem Celular , Cães , Humanos , Ictiose Ligada ao Cromossomo X/enzimologia , Esteril-Sulfatase , Relação Estrutura-AtividadeRESUMO
Steroid hormones, such as glucocorticoids (GC), influence immune and inflammatory responses through their suppressive actions. Recent evidence suggests that another steroid hormone, dehydroepiandrosterone (DHEA), provides an immunostimulatory influence opposing the effect of GC. DHEA circulates in its inactive sulphated form, DHEAS, requiring conversion to DHEA by a steroid sulphatase (SS) enzyme for biological activity. Therefore, inhibition of SS activity may affect immune responses, allowing endogenous GC effects to predominate. We have shown that administration of DHEA and DHEAS in contact sensitization (CS) augments ear swelling by 39 and 46% respectively (P < 0.001). DHEAS at doses of 0.5, 5 and 50 mg/kg reverses the inhibitory effect of corticosterone (5 mg/kg) (P < 0.01). In CS, CT2251 (SS inhibitor) at 10 and 0.1 mg/kg inhibited ear swelling by 61 and 38% (P < 0.05) respectively. In addition, it inhibited DHEAS-augmented responses by 49 and 35% respectively (P < 0.05), with no effect on DHEA-augmented responses. DHEAS reversed CT2251 inhibition of the CS response with complete reversal at 50 mg/kg (P < 0.05). DHEAS and CT2251 appear to affect cellular infiltration into the ear, since DHEAS increased the number of lymphocytes by 63.8% and macrophages by 107% (P < 0.001), whereas CT2251 at 0.1 mg/kg decreased the number of lymphocytes by 65% (P < 0.001) and macrophages by 80% (P < 0.001). DHEAS, CT2251 and dexamethasone had no effect on oedema in the ear. From our data we have shown that steroid hormones, such as DHEA, have the potential to act as immunostimulatory factors in vivo. Inhibiting the conversion of DHEAS to DHEA by SS enzyme leads to an anti-inflammatory effect.
Assuntos
Adjuvantes Imunológicos , Arilsulfatases/fisiologia , Desidroepiandrosterona/imunologia , Dermatite de Contato/imunologia , Animais , Arilsulfatases/antagonistas & inibidores , Sulfato de Desidroepiandrosterona/imunologia , Dermatite de Contato/prevenção & controle , Dexametasona/imunologia , Inibidores Enzimáticos/farmacologia , Estrona/análogos & derivados , Estrona/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxazolona/imunologia , Esteril-SulfataseRESUMO
We have examined the cytolytic and bactericidal activity of resting and cytokine-stimulated natural-killer (NK) cells against K562 and Daudi cell lines and Escherichia coli, respectively. Unstimulated NK cells showed considerable cytolytic activity against K562 (64 +/- 4%) and relatively low activity against the Daudi cell lines (22 +/- 9%). Pretreatment of NK cells with the arylsulfatase (AS) type-II-specific inhibitor NaH2PO4 reduced cytotoxicity towards K562 and Daudi 1.3- and 2.9-fold (p < 0.05; n = 12), respectively, indicating that AS participates in NK-mediated cytotoxicity. Interleukin-2 (IL-2) (200 units/ml) caused a 1.3- and 3.5-fold (p < 0.5; n = 12) enhancement of NK cytotoxic activity against K562 and Daudi, respectively. Pretreatment of these cells with the AS type-II-specific inhibitor NaH2PO4 reduced cytotoxicity 1.1-fold towards K562 (p < 0.05; n = 12) and 1.2-fold towards Daudi (p > 0.05; n = 12) indicating that AS does not participate in IL-2-mediated NK cytolytic activity against these cell lines. IL-7 (3 units/ml) did not cause any enhancement of NK cytolytic activity. Unstimulated NK cells showed considerable bactericidal activity against E. coli (23 +/- 4%). Incubation of resting NK cells with NaH2PO4 reduced the bactericidal effect only by 1.09-fold (p > 0.05; n = 12), indicating that AS does not mediate this effect. IL-2 (200 units/ml) and IL-7 (3 units/ml) enhanced the bactericidal activity 1.5- and 2.2-fold, respectively (p < 0.05; n = 12). This effect was not influenced by incubation of IL-2-stimulated cells with NaH2PO4, indicating that AS does not participate in the IL-2-mediated NK bactericidal effect. IL-2 seems to exert its stimulatory effect upon NK-mediated bactericidal activity by a different, non-AS-dependent mechanism. However, incubation of IL-7-stimulated NK cells with NaH2PO4 reduced the NK bactericidal effect by 1.2-fold (p < 0.05; n = 12), indicating that AS may have a role in this reaction. These data can be further confirmed by detection of AS through degranulation of NK cells, showing that IL-2 induced only mild degranulation of resting and f-MLP-stimulated NK cells (26 +/- 1% vs 22 +/- 2% and 31 +/- 2% vs 29 +/- 2, respectively) (p > 0.05; n = 8). In contrast, IL-7 showed significant enhancement of AS release in resting or f-MLP-induced NK cells (36 +/- 3% vs 22 +/- 2% and 49 +/- 3% vs 29 +/- 2%, respectively) (p < 0.05; n = 8).
Assuntos
Arilsulfatases/fisiologia , Interleucina-2/farmacologia , Interleucina-7/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/fisiologia , Atividade Bactericida do Sangue/efeitos dos fármacos , Degranulação Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Células Matadoras Naturais/citologiaRESUMO
Human chorion and decidua use pregnenolone sulfate (P5S) and dehydroepiandrosterone sulfate (DHAS) as substrates for local estrogen and progesterone synthesis. We hypothesized that the local estrogen/progesterone ratio may influence contractility of the adjacent myometrium and hence effect the timing of parturition. Thus, we studied steroid sulfohydrolase activity for P5S in these tissues and investigated the potential interaction of other steroids on the rates of hydrolysis of P5S and DHAS. The enzyme was present in both tissues, predominantly in the microsomal fraction. With P5S as substrate, the Michaelis-Menten constant (Km) was similar in chorion (1.3 +/- 0.2 mumol/L, mean +/- SEM) and decidua (0.9 +/- 0.1 mumol/L) but the maximum velocity (Vmax) was significantly greater in chorion (2.6 +/- 0.4 vs. 1.1 +/- 0.3 nmol/mg protein/15 min, P less than 0.05). In both tissues there was a tendency towards greater activity in tissues obtained before labor compared to tissues obtained after spontaneous labor onset. Using either DHAS or P5S as substrate, there was significant inhibition of sulfohydrolase activity by other steroids at concentrations similar to those in late pregnancy fetal and maternal plasma. In microsomal preparations using DHAS as substrate, activity was inhibited by equimolar concentrations of estrone sulfate (E1S, by 38 +/- 2%), P5S (by 74 +/- 2%), and cholesterol sulfate (C27S, by 38 +/- 3%). With P5S as substrate, equimolar concentrations of E1S, DHAS, and C27S caused inhibition of sulfohydrolase activity by 19 +/- 5%, 16 +/- 4%, and 18 +/- 2%, respectively. These inhibitory effects also were observed using a tissue explant system with intact cells. In kinetic inhibition studies using DHAS as substrate, E1S and P5S were competitive inhibitors with inhibition constants (Ki) of 4.8 +/- 1.3 and 0.7 +/- 0.1 mumol/L, respectively. Using P5S as substrate, E1S and DHAS also were competitive inhibitors with Ki values of 8.2 +/- 2.1 and 9.6 +/- 1.2 mumol/L, respectively. For both substrates, the pattern of inhibition by C27S was complex. Preliminary experiments to distinguish, on the basis of differing physical-chemical properties, separate enzymes for different substrates were inconclusive. We conclude that human chorion and decidua can hydrolyze several steroid sulfoconjugates and this activity may regulate local estrogen and progesterone synthesis. There are significant interactions among steroid sulfoconjugates in regulating this activity. These activities may be important components of a paracrine system that determines myometrial contractility and the timing of parturition.
Assuntos
Arilsulfatases/fisiologia , Córion/enzimologia , Decídua/enzimologia , Desidroepiandrosterona/metabolismo , Pregnenolona/metabolismo , Sulfatases/fisiologia , Técnicas de Cultura , Desidroepiandrosterona/farmacocinética , Estrogênios/biossíntese , Estrona/análogos & derivados , Estrona/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Trabalho de Parto/metabolismo , Microssomos/metabolismo , Gravidez , Pregnenolona/farmacocinética , Progesterona/biossíntese , Esteril-Sulfatase , TemperaturaRESUMO
Arylsulphatase and acid phosphatase activity were demonstrated cytochemically in spermatozoa of the marine mussel Mytilus edulis. Reaction product resulting from arylsulphatase activity was measured using an integrating microdensitometer and found to increase with incubation time and to be variable according to the pH of the incubation medium. Two peaks in activity, at pH 4.5 and 6.0 were evident for some experimental protocols suggesting the possibility of two isoenzymes; however, studies on the ultrastructural localization of the enzyme showed no difference between sites of activity for the two pH values. Ultrastructural localization of arylsulphatase showed activity associated with the Golgi body of developing spermatids and in particular within the proacrosomal vesicles but limited to the periphery of the acrosomal vesicle which is formed with the fusion of the proacrosomal vesicles. In spawned spermatozoa arylsulphatase activity was localized in association with the axial rod and subacrosomal material; activity also occurred along the outer acrosomal membrane and within the acrosomal vesicle and also associated with the acrosomal process following the acrosome reaction. Sulphate groups were demonstrated cytochemically within the vitelline coat of oocytes in the mantle tissue. These findings suggest that arylsulphatase could be one of the lysins previously demonstrated in M. edulis spermatozoa. Acid phosphatase activity was demonstrated in spawned spermatozoa around the nuclear envelope and along the outer acrosomal membrane.