Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
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A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.
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PMID:Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U). 1270 44

A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.
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PMID:Isolation and characterization of gelatin-binding proteins from goat seminal plasma. 1273 34

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.
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PMID:Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins. 1458 8

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [high-activity PLTP (HA-PLTP)] and the other a low-activity (LA-PLTP) form. We present here a PLTP ELISA that allows not only for accurate measurement of PLTP concentration in plasma but also of the distribution of both LA- and HA-PLTP. To achieve similar immunoreactivity of the two PLTP forms, a denaturing sample pretreatment with 0.5% SDS was required. Distribution of LA- and HA-PLTP in plasma was assessed using size-exclusion chromatography, Heparin-Sepharose chromatography, anti-PLTP immunoaffinity chromatography, and dextran sulfate-CaCl2 precipitation. All four methods demonstrated that approximately 60% of plasma PLTP represents LA-PLTP and 40% represents HA-PLTP. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n = 80) was 5.81 +/- 1.33 mg/l (mean +/- SD) (range, 2.78-10.06 mg/l) and the mean activity was 5.84 +/- 1.39 micromol/ml/h (range, 3.21-11.15 micromol/ml/h). To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl2 precipitation with the modified PLTP ELISA. The HA-PLTP mass (mean, 1.87 +/- 0.85 mg/l) correlated significantly with serum PLTP activity, whereas that of LA-PLTP (mean, 3.94 +/- 1.4 mg/l) showed no correlation with phospholipid transfer activity.
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PMID:Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA. 1461 37

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the smooth muscle. MMP-2 has been purified from plasma membrane fraction of bovine pulmonary artery smooth muscle to homogeneity using a combination of purification steps. Heparin sepharose purified preparation of 72 kDa progelatinase is composed of two distinct population of zymogens: a 72 kDa progelatinase tightly complexed with TIMP-2 (an ambient tissue inhibitor of metalloprotease in the smooth muscle plasma membrane), and a native 72 kDa progelatinase free of any detectable TIMP-2. The homogeneity of the native 72 kDa progelatinase form is demonstrated by SDS-PAGE under non-reducing condition, non-denaturing native gel electrophoresis. The purified TIMP-2 free proenzyme electrophoresed as a single band of 72 kDa which could be activated by APMA with the formation of 62 and 45 kDa active species. The proenzyme is activated poorly by trypsin but not by plasmin. The purified 72 kDa progelatinase is stable at aqueous solution and does not spontaneously autoactivate. The purified 72 kDa gelatinase exhibited properties that are typical of MMP-2 obtained from other sources. These are: (i) its activity is dependent on the divalent cation, Ca+2, and is inhibited by EDTA, EGTA and 1:1 0-phenanthroline; (ii) it was inhibited by a, macroglobulin but not by the inhibitors of serine, cysteine, thiol, aspartic proteinases and calpains; (iii) it was found to be inhibited by TIMP-2, the specific inhibitor of MMP-2; (iv) like MMP-2, obtained from other sources, its major substrates were found to be collagens (type IV and V) and gelatins (type I, IV and V). Additionally, the purified MMP-2 degrades Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH (dinitrophenyl labelled peptide), a well known synthetic substrate for the MMP-2.
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PMID:Identification, purification and characterization of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle plasma membrane. 1503 Jan 72

The interaction of boar seminal plasma proteins and sperm with yeast mannan was investigated by the enzyme-linked binding assay (ELBA) and specific detection of proteins after SDS electrophoresis and blotting using biotinylated derivative of the polysaccharide. Heparin-binding proteins (especially AQN 1 and DQH proteins) and their aggregated forms showed affinity to yeast mannan. Besides that, these proteins were shown to bind to oviductal epithelium. The mannan-binding activity of boar proteins and sperm was inhibited most efficiently by ovomucoid, ovalbumin and N-glycans released from ovalbumin, but not with d-glucose, d-mannose and their phosphates. On the other hand, yeast mannan inhibited both the interaction of boar seminal plasma and sperm with heparin and the binding of these proteins to porcine oviductal epithelium. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. Proteins adsorbed to the immobilized polysaccharide were analyzed by RP-HPLC, SDS electrophoresis and N-terminal amino acid sequencing. AQN and AWN spermadhesins and DQH protein (names are derived from the N-terminal amino acid sequence) were identified as components of the isolated fraction. The results suggest an involvement of mannan-binding proteins in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both the complex d-mannose-containing saccharide structures and the heparin may also play an important role in sperm release from the oviductal reservoir or the capacitation process.
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PMID:Mannan-binding proteins from boar seminal plasma. 1528 92

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.
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PMID:Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane. 1537 20

An endonuclease was isolated from 5 days old Agropyron elongatum 8x = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme's moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn(2+) and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI > or = polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow C bond. The substrate's products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3' position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.
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PMID:Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings. 1559 99

Investigations on specific and functionally active sperm antigens would bring about the elucidation of the mechanisms of gamete interaction and help the search to new approaches for prognosis and regulation of fertility. Previously, we have produced a polyclonal rabbit anti-boar spermatozoa antibody (RABSA) that might affect the fertilizing capacity of boar spermatozoa. The sperm specificity of RABSA was demonstrated by double immunodiffusion, immunoelectrophoresis and ELISA against boar spermatozoa, as well as against saline extracts of boar reproductive and somatic organs. Using indirect immunofluorescence (IIF) test, here we provide evidence that RABSA stained the acrosomes of ejaculated and capacitated boar and human spermatozoa, the fluorescence being intensified on the equatorial region after the acrosome reaction. The RABSA cognate antigen/s is a subject of interest because of their specific localization in sperm structures, which is shown to be a binding and/or fusion competence region. Using ion-exchange (Heparin-Sepharose) chromatography, we eluted an antigen with molecular mass 60 kDa (Ag60) in SDS-PAGE from NP40 extracts of capacitated boar spermatozoa. In Western blot, RABSA recognized specifically this antigen. The Ag60 did not affect the sperm-ligand activity of zona pellucida in a porcine sperm-zona binding assay. IIF experiments showed that zona-free porcine oocytes preincubated with Ag60 and RABSA presented fluorescent labeling over the entire egg surface. The biological and IIF experiments provide evidence supporting the involvement of Ag60 in functional steps required for sperm-egg binding and/or fusion, but not sperm-zona pellucida binding.
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PMID:Identification and isolation of boar sperm specific antigens with potential role in sperm-egg interaction. 1559 29

Arylsulfatase cloned from a marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, was overexpressed in Escherichia coli with 10 microM IPTG induction. The expressed recombinant arylsulfatase was purified to homogeneity from the harvested cells through osmotic disruption and column chromatography methods, such as DEAE-cellulose anion exchange chromatography and Heparin-Sepharose affinity chromatography. The purified arylsulfatase was kinetically characterized using the synthetic substrate of phenolic ester, p-nitrophenyl sulfate (pNPS). One unit of arylsulfatase catalyzes the liberation of 1.0 micromol p-nitrophenol from pNPS per minute. The purified enzyme has a specific activity of 468 U/mg with a purification yield of 27% from the cell lysate, and exhibited an estimated molecular mass of 33 kDa in SDS-PAGE analysis. The precursor polypeptide of 36 kDa was processed by releasing a putative signal peptide, and the mature arylsulfatase of 33.1 kDa with a N-terminal sequence of S-E-T-K-N was trafficked to periplasmic space. The enzyme had optimum reaction conditions for activity at pH 7.0 and at a temperature range of 40-45 degrees C. The apparent K(M) and k(cat) of the enzyme for hydrolysis of pNPS at pH 7.0 and at 45 degrees C were determined to be 1.15 mM and 1000 s-1, respectively. Based on inhibitor studies along with optimal pH values and preferential periplasmic location of the enzyme, we suggest that the recombinant arylsulfatase from P. carrageenovora is probably similar to the Klebsiella sulfatase with serine residue in the active site.
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PMID:Purification and characterization of the recombinant arylsulfatase cloned from Pseudoalteromonas carrageenovora. 1559 66


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