Acid sphingomyelinase from human urine: purification and characterization

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).     

Heterogeneity of ovine prolactin after labeling with iodine 125: value of labeling with lactoperoxidase]

The authors have shown an heterogeneity of the ovine prolactine molecule after labelling with iodine 125. As well with chloramine T as with lactoperoxydase, it appears three molecular species which react with the immune serum antiprolactine (I.S.). The first species is of high molecular weight and is probably constituted of aggregates. Their combination with the I.S. is non specific and give blanks of high value. The second species is a dimere and the third one is the monomere. The two last species react with the I.S. and can give competition curves when they are choosen as tracer. However, if one uses as tracer a product obtained by labelling with chloramine T, the competition appears for high concentrations of native hormone. As if the I.S. recognizes much more the labelled protein than the native one. But if one uses the same species but labelled with lactoperoxydase, the competition appears for concentrations lower than five nanogrammes. In the same time one can see that the specificity toward different I.S. is modified. The authors think that the labelling with lactoperoxydase better preserves the tertiary structure of the native protein than do the labelling with chloramine T.     

Partial purification and properties of acid sphingomyelinase from rat liver

Acid sphingomyelinase was purified approximately 5,200-fold from the mitochondria-lysosome-enriched particles of rat liver by sequential chromatography on DEAE-cellulose, octyl-Sepharose, Sephacryl S-300, Concanavalin A-Sepharose, and CM-cellulose. The specific activity of this highly purified enzyme was 3.2 mmol per hr per mg protein. The enzyme was active against 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, but bis-4-methylumbelliferyl-phosphate and bis-p-nitrophenyl-phosphate were poor substrates. The preparation was free of Mg2+-dependent neutral sphingomyelinase and eight lysosomal enzymes except for the trace amount of acid phosphatase and beta-galactosidase. Apparent molecular weight of the enzyme was 200,000, estimated by Sephadex G-200 filtration in 0.1% Triton X-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed three major bands corresponding to molecular weights of 45,600, 44,500, and 40,000 with several minor bands. Characterization of the enzyme revealed almost the same properties as those of human tissues reported by other investigators, including pH optimum, requirement of Triton X-100, effects of metal divalent cations, phosphate ion, EDTA, some thiol blocking reagents, and amphophilic drugs.     

Acid sphingomyelinase, cell membranes and human disease: Lessons from Niemann-Pick disease

Acid sphingomyelinase (ASM) plays an important role in normal membrane turnover through the hydrolysis of sphingomyelin, and is one of the key enzymes responsible for the production of ceramide. ASM activity is deficient in the genetic disorder Types A and B Niemann-Pick disease (NPD). ASM knockout (ASMKO) mice were originally constructed to study this disorder, and numerous defects in ceramide-related signaling have been shown. Studies in these mice have further suggested that ASM may be involved in the pathogenesis of several common diseases through the reorganization of membrane microdomains. This review will focus on the role of ASM in membrane biology, with a specific emphasis on what a rare genetic disorder (NPD) has taught us about more common events.     

Identification,partial purification,and localization of a neutral sphingomyelinase in rabbit skeletal muscle: Neutral sphingomyelinase in skeletal muscle

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic -cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).     

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.     

The purification and identification of calmodulin from human placenta

A protein which showed similarity to bovine brain calmodulin in electrophoretic mobilities on polyacrylamide gels in the presence of 40% glycerol (pH 8.6) and 0.1% sodium dodecyl sulfate (pH 7.2) was isolated from human placenta. Its final yield was approx. 4 mg per kg human placenta. The placenta protein was similar to bovine brain calmodulin in stimulating bovine brain calmodulin-deficient cyclic nucleotide phosphodiesterase in the presence of calcium. However, its stimulating activity was eliminated by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or trifluoperazine. In addition, there is a close resemblance in amino acid composition between the placental protein and bovine brain calmodulin. These results indicate that calmodulin is present in human placenta.     

DNA-binding proteins of human placenta: purification and characterization of an endonuclease

DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125–0.15M potassium phosphate. PC fractions containing electrophoretically pure 34KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3–5 mM Mg²⁺ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate.     

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.     

Endogenous heparin-binding lectin activity in human placenta: purification and developmental expression

Human placental extracts contain a herapin-inhibitable lectin activity. The lectin, which closely resembles those from chicken and rat tissues, was purified by heparin-affinity chromatography. It shares many properties with the previously reported lectins, including hapten specificity, molecular weight of monomers, and immunological cross-reactivity. Sections from different stages of placental development, stained by immunohistochemistry procedures using lectin-specific antibody, showed that the lectin was initially present only in cytotrophoblasts of early first trimester villi. Later in the first trimester, both cytotrophoblasts and syncytiotrophoblasts were stained positively for lectin. From second trimester to term, the lectin was seen only in syncytiotrophoblasts.     

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.     

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.     

Expression and purification of human placenta lactogen in Escherichia coli

There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quantities of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E. coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 degrees C. Determination of the yield of recombinant hPL by SDS-PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8M urea and purified by a His6 tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL.     

15-ketoprostaglandin delta13 reductase from human placenta: purification, kinetics, and inhibitor binding.

An NADH-dependent 15-ketoprostaglandin Δ¹³ reductase has been purified to near homogeneity from human placenta by a procedure which includes affinity chromatography on blue Sepharose. The enzyme utilizes as substrates 15-ketoprostaglandins of the E, F, A, and B series, and the reaction is experimentally irreversible. Molecular weight estimations on Sephadex G-100 and sodium dodecyl sulfate disc gel electrophoresis suggest that the enzyme is a dimer. The subunits appear to be similar in size if not identical and have a molecular weight of 35,000. The mechanism of the reaction of 15-ketoprostaglandin E₂ and NADH catalyzed by this enzyme has been investigated by steady-state kinetic methods. The 13,14-dihydro-15-ketoprostaglandin product is an inhibitor of the reaction, being competitive with respect to 15-ketoprostaglandin E₂ and noncompetitive with respect to NADH; NAD⁺ does not inhibit the reaction. NADPH and Cibacron blue 3G-A are “dead-end” inhibitors of the reaction; both act competitively with respect to NADH and noncompetitively with respect to 15-ketoprostaglandin E₂. These observations are consistent with a rapid equilibrium random mechanism with the formation of an unreactive enzyme · NADH · 13,14-dihydro-15-ketoprostaglandin E₂ complex. The interaction of NADPH and Cibacron blue 3G-A with the free enzyme was investigated further by fluorimetry. Both substances bind to the free enzyme and quench its fluorescence. This property was utilized to titrate the enzyme, and a value of 3.28 × 10^?11 mol of binding sites/mU of enzyme was obtained.