The intracellular distribution of arylsulphatase C and oestrogen sulphatase activities in rat kidney and spleen.
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ABSTRACT:
SUBMITTER: Thomas SB
PROVIDER: S-EPMC1164018 | biostudies-other | 1976 Sep
REPOSITORIES: biostudies-other
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Project description:Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.
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Project description:1. The properties of enzyme activities hydrolysing the sulphate esters of dehydroepiandrosterone, oestrone and p-nitrophenol are reported. The preparation studied was obtained from the microsomal fraction of human placenta by ultrasonic treatment and addition of Triton X-100. 2. The behaviour of the preparation during sedimentation at 105000g and attempts at purification indicated that the activities were particulate. Electron microscopy demonstrated the rupture of vesicular structures approx. 0.5mu in diameter concurrent with the release of activity. 3. The three activities were always associated throughout repeated attempts at separation by sucrose-density-gradient centrifugation and Sephadex-gel filtration. On the basis of kinetic studies, stability studies and treatment with butanol and ribonuclease it was concluded that a separate enzyme is responsible for each of the three activities. Widely varying plots of activity as a function of pH were consistent with this conclusion. 4. On the basis of sensitivity of the enzymes hydrolysing dehydroepiandrosterone sulphate and oestrone sulphate to ribonuclease and sensitivity of all three enzymes to lipase, it was concluded that the three enzymes are bound to a particle containing lipid and RNA. Enzymic activity is dependent on structural integrity of the particle. 5. A spectrophotometric method for the assay of oestrone sulphate hydrolysis is described. 6. Hydrolysis of nitrocatechol sulphate by human placenta under conditions described for arylsulphatases A and B is reported.
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Project description:Sulphatases undergo a unique post-translational modification that converts a highly conserved cysteine located within their active site into formylglycine. This modification is necessary for the catalytic activities of the sulphatases, and it is generated by the protein product of sulphatase-modifying factor 1 (SUMF1), the gene mutated in multiple sulphatase deficiency (MSD). A paralogous gene, SUMF2, was discovered through its sequence similarity to SUMF1. We present evidence that SUMF2 colocalizes with SUMF1 within the endoplasmic reticulum and that the two proteins form heterodimers. SUMF1 and SUMF2 also form homodimers. In addition, SUMF2 is able to associate with the sulphatases with and without SUMF1. We have previously shown that co-transfection of SUMF1 with the sulphatase complementary DNAs greatly enhances the activities of the overexpressed sulphatases. Here, we show that SUMF2 inhibits the enhancing effects of SUMF1 on sulphatases, suggesting that the SUMF1-SUMF2 interaction represents a further level of control of these sulphatase activities.
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Project description:BackgroundSteroid sulphatase (STS) is one of the steroid-metabolising enzymes involved in desulphating inactive steroid sulphates and oestrogen sulphotransferase (EST) sulphates active oestrogen. The roles of both STS and EST have not been examined in oestrogen-dependent non-small-cell lung cancer (NSCLC).MethodsWe evaluated the immunoreactivity of STS and EST in NSCLC cases using immunohistochemistry. The function of STS and EST was further demonstrated using NSCLC cell lines.ResultsThe immunoreactivity of STS and EST was detected in 49.5% and 27.8% of NSCLC cases, respectively. The immunoreactivity of STS was significantly higher in female adenocarcinoma cases. The STS-positive NSCLCs were also significantly correlated in an inversed manner with tumour size and cell proliferation and tended to be associated with better clinical outcome. However, the immunoreactivity of EST was significantly correlated with intracellular oestradiol concentration. Results of in vitro analysis demonstrated that oestrone sulphate (E1-S) induced and pregnenolone sulphate (Preg-S) inhibited the proliferation in STS-expressing cell lines. The inhibition by Preg-S was reversed by a specific progesterone receptor blocker. Simultaneous addition of E1-S and Preg-S significantly suppressed the proliferation.ConclusionIn NSCLC patients, STS is considered a good prognostic factor. Results of our present study also indicated the benefits of potential progesterone therapy for NSCLC patients.
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Project description:A sulphamidase activity present in rat spleen capable of hydrolysing N-[(35)S]sulphated heparin was studied. This activity was associated with the lysosomal fraction. Studies in vivo showed that the rat is capable of significantly desulphating heparin. Lysosomes in all the major tissues can effectively accumulate heparin. The heparin sulphamidase and arylsulphatase activities from rat spleen were separated by isoelectric focusing. Heparin sulphamidase was a distinct entity from all the arylsulphatase activities.
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Project description:1. A technique for perfusing the isolated rat kidney is described. It is primarily designed for the study of renal metabolism but is also suitable for studying some aspects of the secretory function; this was normal with respect to minimal glucosuria. The glomerular filtration rate as measured by creatinine clearance was lower than in vivo and slowly decreased with time. 2. Gluconeogenesis from a variety of precursors was rapid and similar to that in kidney-cortex slices, in contrast with liver where the perfused organ is more effective than slices. Whereas the maximal rates of gluconeogenesis from glycerol and pyruvate were similar in liver and kidney, the rates from succinate, malate and fumarate were 14-20 times, and those from glutamate and aspartate about three times, as high in the kidney. 3. The oxygen consumption of the perfused organ was about twice that of cortex slices, presumably because of the secretory work done in the perfused organ but not in slices. 4. The rate of acetoacetate oxidation was about the same in the perfused organ and in slices but, because of the higher rate of oxygen consumption, the percentage contribution of acetoacetate to the fuel of respiration was lower in the perfused organ. The results suggest that acetoacetate can supply energy for the basal requirements and for gluconeogenesis but not for the secretory work. 5. Glutamine was formed at a high rate from glutamate and at a lower rate from aspartate. The high rates indicate that, in the rat, the kidney is a major source of body glutamine.
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Project description:1. Free and total activities of beta-glucosidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and beta-glucuronidase have been determined fluorimetrically in five subcellular fractions of rat kidney. 2. The beta-glucosidase activity appeared in the soluble fraction, beta-glucuronidase had the distribution pattern of a lysosomal enzyme, and both beta-galactosidase and N-acetyl-beta-glucosaminidase had bimodal distributions. 3. Two types of beta-galactosidase activity were found: a sedimentable type, having optimum pH3.7, mol.wt. about 80000 and slow electrophoretic mobility at pH7.0 in starch gel; and a soluble type of much faster mobility, having optimum pH5.5-6.5 and mol.wt. about 40000. 4. Evidence is presented that the beta-glucosidase and the soluble type of beta-galactosidase are the same enzyme. 5. Most of the N-acetyl-beta-glucosaminidase activity was in the lysosome-rich fractions, but a significant proportion occurred in the microsomal fraction in a non-latent form. 6. The use of beta-galactosidase and N-acetyl-beta-glucosaminidase as lysosomal marker enzymes is complicated by the possible presence of multiple forms, but this limitation does not apply to beta-glucuronidase in the rat kidney.
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Project description:Sample preparation optimisation for extracting in a quantitative manner proteins from rat spleen
2016-07-04 | PXD003520 | Pride
Project description:The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.
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