Estrogen sulfotransferase in human placenta

Human placental estrogen sulfotransferase (ESFT) was partially purified from the term placental cytosol by (NH4)2SO4 precipitation and agarose gel chromatography. Additional purifications caused a rapid loss of the enzyme activity. The activity was abolished by isoelectrofocusing but partially retained by chromatofocusing. The value of pI of human placental ESFT is 5.8 and the same value was obtained for bovine adrenal ESFT. The enzyme protein was able to bind to the affinity resin, estradiol-17-hemisuccinyl-1,2-diaminododecane sepharose 4B, but difficult to be extracted by estradiol (E2). The extract of the affinity resin showed one major protein band at 68,000 dalton on SDS-polyacrylamide gel electrophoresis. Kinetic studies using partially purified ESFT revealed that E2 is the best substrate for this enzyme. The relative rate of sulfurylation of E2, estrone, estriol and dehydroepiandrosterone at 4 microM (Km for E2) is 1, 0.3, 0.08 and 0.08, respectively.     

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl2 or CaCl2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52,300 and 50,000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26- and 90-fold for liver and chorion, respectively. Both enzymes were eluted from agarose-hexane-adenosine 3',5'-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.     

Solubilization and partial purification of steroid sulfatase of human placenta

Steroid sulfatase of human placenta has been solubilized by treatment of the microsomal fraction with an amphoteric surface active agent, Miranol H2M and ultrasound. Criteria of solubility include non-sedimentation of the activity following centrifugation at 160,000 × g, its retention on Sepharose 6B and a single peak of activity after polyacrylamide gel electrophoresis. Enzyme activity was located in the same gel fractions for the two substrates tested; cholesterol sulfate and dehydroisoandrosterone sulfate. The addition of dithiothreitol was found necessary to maintain the stability of the enzyme indicating the presence of sulfhydryl groups in the molecule. A molecular weight of approximately 330,000 has been estimated from the elution volume of the enzyme system on a column of Sepharose 6B. It is believed that this protein represents a sulfatase enzyme complex composed of subunits with different specificities. From kinetic studies, a K_m of 6.2 × 10^?5M for the cleavage of dehydroisoandrosterone sulfate and a K_m of 2 × 10^?6M for the cleavage of cholesterol sulfate have been calculated.     

Estrogen sulfotransferase distribution in tissues of mouse and guinea pig: steroidal inhibition of the guinea pig enzyme.

The highest total activity of estrogen sulfotransferase in guinea pig is in liver and the highest specific activities are in the adrenal and the midgestational chorion. Guinea pig gonads contain scarcely detectable activities. In CD-1 mice the highest specific activity is in testis and the highest total activity is in late placenta. Adrenals from both sexes and ovaries contain minimal activities, while liver and fetal membrane activities are remarkably low. In CD-1, DBA, C57BL, and BALB mice, qualitative patterns are similar. Purified or partially purified estrogen sulfotransferase from guinea pig adrenal and chorion were used to study the effect of a number of possible steroidal inhibitors. Considerable structural specificity is evident within a range of steroids, even among some which are not substrates. Pregnenolone is the most effective 21-carbon inhibitor and, in general, more highly hydroxylated forms are less inhibitory. Within a series of 21-, 19- and 18-carbon steroids, the structure of the A ring appears to be extremely important in regard to inhibitory effects.     

N-acetylgalactosamine-6-sulfate sulfatase in human placenta: purification and characteristics.

N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.     

Extraction and partial purification of mouse uterine alkaline phosphatase.

Alkaline phosphatase in uterine homogenates from day 7 pregnant mice was solubilized using 0.2% (v/v) Triton X-100 and extracted wtih 20% (v/v) n-butanol. The procedure, which resulted in 182-fold purification, included ammonium sulfate precipitation, DEAE-cellulose anion exchange chromatography and Sephadex G200 gel filtration. Solubilization with Triton X-100 was an important step in the procedure since extraction with n-butanol alone only partially solubilized the enzyme and gave low extraction yields, much of the enzyme activity remaining in association with negatively charged residues. However, butanol extraction of Triton X-100-treated homogenates gave high yields of enzyme and eliminated p-nitrophenyl phosphatases which displayed activity in the pH range 3.0--7.5, together with a large proportion of inactive protein. The activity of the purified enzyme preparations was electrophoretically homogeneous on cellulose acetate membranes, suggesting that the alkaline phosphatase in the mouse uterus exists in a single isozymic form. Polyacrylamide-gel electrophoresis revealed that the purified preparations contained at least one protein as an impurity. Attempts to further purify the alkaline phosphatase by isoelectric focusing were unsuccessful since the enzyme was found to have an isoelectric point of about 5.0 and at this pH it was rapidly inactivated.     

Estrogen sulfotransferase activity in guinea pig uterus and chorion

An estrogen sulfotransferase (ST) is detectable in high speed supernatants of pregnant guinea-pig uterus and shows maximum activity between about 47 and 55 days of gestation, with a decrease toward term. No appreciable activity was apparent in the non-pregnant state or before at least 43 days of pregnancy. A considerably higher ST activity is present in chorion as early as 30 days of gestation, and this also decreases toward term. The two ST's exhibit similar KM (0.1-0.13 microM with estrone as substrate) and pI (5.8) values, as well as similar specificities. Estradiol-17 beta and estriol are sulfurylated 82 and 6% that of estrone at equimolar concn. Neither p-nitrophenol nor several neutral steroids are substrates for the enzymes. Enzyme activity is poorly expressed in the absence of thiol groups, the presence of monothioglycerol stimulating uterine and chorion enzymes by 5- and 15-fold, respectively. Stimulation is also observed in the presence of Mg2+, Ca2+ or Mn2+. Chromatofocusing on a poly buffer ion-exchanger from pH 7.4 to 4.0 resulted in elution of a sharp peak of enzyme activity, at pH = 5.8, from both tissues provided that the eluting buffer contained thiol groups and 0.25 M sucrose. This single step resulted in at least a 35- to 100-fold increase in specific activity. The partially purified enzyme from chorion exhibited a KM for estrone of 0.13 microM.     

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

Behavior of mouse placental and uterine estrogen sulfotransferase during chromatography and other procedures

The estrogen sulfotransferase activity of high-speed supernatants of mouse placenta and uterus behaves on conventional and high-performance liquid chromatographic gel filtration as an enzyme species with a molecular weight of the order of 30 000. This is so whether the cytosols are freshly prepared or have been stored at -20 degrees C before chromatography. The presence of thiol groups or EDTA has no effect on the elution pattern. The partially purified enzyme is extremely unstable and is poorly recovered by (NH4)2SO4 fractionation. Some stabilization can be achieved in the presence of 0.1 microM estradiol. Chromatofocusing of cytosols results in the elution of one or two sulfotransferase peaks, depending upon experimental conditions such as the presence or absence of thiol groups. These peaks act upon both estrone and estradiol as substrates. Chromatofocusing by fast protein liquid chromatography (FPLC) in the absence of thiol groups results in the elution of one sulfotransferase peak whose activity can be detected only when thiol groups are present during enzyme assay.     

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.     

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...     

The partial purification of growth-inhibiting factor of the brachypodism-H mouse embryos

The extract of 13-day-old $\text{bp}{}^{\mathrm{H}}\text{bp}{}^{\mathrm{H}}$ embryos contains the factor causing inhibition of the growth of the long bone rudiments of normal embryos in vitro. A 400-fold purification of the factor has been achieved. The molecular weight of the main protein component of the preparation obtained is 76,000. The synthesis of this protein appears to be controlled by bp^H gene.     

Isolation, partial purification, and characterization of a novel petromyzonol sulfotransferase from Petromyzon marinus (lamprey) larval liver

We have isolated, partially purified, and characterized the 5 alpha-petromyzonol (5 alpha-PZ), (5 alpha-cholan- 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-) sulfotransferase (PZ-SULT) from larval lamprey liver. Crude liver extracts exhibited a PZ-SULT activity of 0.9120 pmol/min/mg in juvenile and 12.62 pmol/min/mg in larvae. Using crude larval liver extracts and various 5 beta-cholan substrates and allocholic acid there was negligible activity, however, with 5 alpha-PZ and 3-keto-5 alpha-PZ the SULT activity was 231.5 pmol/min/mg and 180.8 pmol/min/mg respectively. This established that the sulfotransferase of lamprey larval liver extracts prefers (5 alpha) substrates and it is selective for hydroxyl at C-24. PZ-SULT was purified through various chromatography procedures. Partially purified PZ-SULT exhibited a pH optimum of 8.0, a temperature optimum of 22 degrees C, and activity was linear for 1h. PZ-SULT exhibited a K(m) of 2.5 microM for PAPS and a K(m) of 8 microM for PZ. The affinity purified peak PZ-SULT exhibited a specific activity of 2,038 pmol/min/mg. The peak protein upon SDS-PAGE, correlated to an Mw 47 kDa. Photoaffinity labeling with PAP(35)S, specifically crosslinked the 47 kDa protein, further confirming the identity of PZ-SULT. Partial amino acid sequencing of the putative 47 kDa PZ-SULT protein yielded a peptide sequence (M)SISQAVDAAFXEI, which possessed an overall (approximately 35-40%) homology with mammalian SULT2B1a.     

Oestrogen sulfotransferase: isolation of a high specific activity species from bovine placenta

During the course of a study of the control of expression of steroid-binding proteins in human mammary cancer oestrogen sulfotransferase was isolated from bovine placenta. By a combination of salt precipitation and ion-exchange and gel-permeation chromatography two forms of the enzyme were isolated. The forms, which apparently differ only in charge, have specific activities 100-300 times greater than has previously been reported for the enzyme. Partial peptide sequences of these enzymes are presented.