Purification and properties of arylsulphatase A from chicken brain

1. Chicken brain arylsulphatase A was purified 2000-fold, with overall recovery 14%, by using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and DEAE-Sephadex column chromatography. 2. The purified preparation was free from beta-glucuronidase, beta-galactosidase, acid phosphatase, inorganic pyrophosphatase and adenosine 3'-phosphate 5'-sulphatophosphate sulphohydrolase activities. 3. Polyacrylamide-gel electrophoresis indicated that the purified preparation was not homogeneous. 4. Chicken brain arylsulphatase was markedly inhibited by carbonyl reagents in the presence of traces of Cu(2+) in the system. Other metal ions such as Fe(2+) and Zn(2+), were inactive. 5. Ascorbic acid alone had no effect on enzyme activity but enhances the inhibition by Cu(2+). 6. Chicken brain arylsulphatase A resembled arylsulphatase A of other animal species in its kinetic properties such as K(m) value, anomalous time-activity relationship and the inhibitory effect of phosphate, sulphite and sulphate ions. However, its electrophoretic mobility, behaviour under zinc acetate fractionation and stimulation by Ag(+) were similar to arylsulphatase B of other animal species. Thus, this enzyme did not correspond to either arylsulphatase A or arylsulphatase B but properties of both. 7. The purified enzyme preparation can degrade cerebroside 3-sulphate.     

Purification and properties of arylsulphatase A from rabbit testis.

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.     

Respiratory inhibition of Sphaerotilus by potassium ferrate

Potassium ferrate (K₂FeO₄) is a strong oxidant that might replace chlorine in the disinfection of water. K₂FeO₄ strongly inhibited the respiration of the bacterium Sphaerotilus, which frequently causes filamentous bulking in activated sludge. To identify the mechanism by which K₂FeO₄ caused inhibition, the distribution of iron sorbed by the bacterium was investigated by a modification of the method of Romano and Peloquin. Iron that penetrated into the cells inhibited the endogenous respiration of Sphaerotilus. The inhibition of the dehydrogenase activity of the bacterium by the ferrate was then studied. This enzyme activity was strongly inhibited by K₂FeO₄, but was restored by the addition of 2-mercaptoethanol. Lineweaver-Burk plots showed that K₂FeO₄ was a non-competitive inhibitor of the respiration of Sphaerotilus.     

Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans

Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11alpha position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11alpha-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c(3+). Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.     

Evaluation of the properties of potassium ferrate used for water purification by luminescence bioassay

Characteristics of four natural water samples from urban and rural areas and the efficiency of a new purifying agent, potassium ferrate K₂FeO₄, were studied by bacterial luminescence bioassay for 30 minutes. It was revealed that two samples of water from the urban areas are toxic, while the other two samples (one from urban and one from rural environment) are nontoxic. Numerous data obtained on the increase in toxicity index with time allow reasonable conclusions to be made about the chemical nature of substances present in the test water samples. Toxic natural water samples were likely to contain heavy metals and were well purified using potassium ferrate, including via their adsorption. In nontoxic natural water samples, toxic complexes with organic compounds present in water could form at the addition of potassium ferrate. The obtained data call for further studying the properties of potassium ferrate complexes with organic compounds. Bacterial luminescence bioassay is a promising method for the rapid assessment of properties of various water sources (their integral toxicity and presumable chemical composition) and new reagents for their purification (effective concentrations, bactericidal properties, and mechanisms of interacting with heavy metals and organic substances in water).     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Modification of human prostatic acid phosphatase by potassium ferrate

Prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase, acid optimum, EC 3.1.3.2) reacts with potassium ferrate, K₂FeO₄ a potent oxidizing agent and an analogue of orthophosphate. Treatment of the enzyme with 10^?6m ferrate at pH 7.5 0 C leads to the immediate loss of 95% of the activity. Molybdate, the competitive inhibitor of prostatic phosphatase, partially protects the enzyme from inactivation. Ferrate inactivation at pH 7.5 is accompanied by the modification of 2 histidine, 4 lysine and 4 methionine residues. Histidine is protected by molybdate, whereas methionine is not and lysine is partly protected. Partial inactivation with ferrate leads to the retardation of the modified enzyme on Sephadex G-200 column, which is eluted in the position of the active monomeric unit.     

高铁酸钾氧化去除饮用水中微量苯酚的研究

采用次氯酸盐氧化法合成高铁酸钾,测试了其对水中苯酚的氧化去除效果及其影响因素.结果表明,苯酚的去除效率主要取决于高铁酸钾和苯酚的质量比.在酸性条件下高铁酸钾对苯酚的去除效果较好.高铁酸钾与苯酚反应主要发生在最初的几分钟时间内,是一个由快而慢的过程.     

Purification and properties of human placenta arylsulphatase A.

1. Arylsulphatase A (arylsulphate sulphohydrolase E.N. 3.1.6.1) has been purified 7200-fold from human placenta using concanavalin A Sepharose chromatography. 2. Ultracentrifugation studies indicated that the purified enzyme was homogenous with respect to sedimentation coefficient and molecular weight and has a molecular weight of 102000. 3. The purified enzyme could hydrolyze cerebroside 3-sulphate, seminolipid and sulphogalactosylsphingosine under identical conditions. 4. The kinetic parameters for the hydrolysis of all sulphate esters used in the present study were the same. 5. Both seminolipid and sulphogalactosylsphingosine were competitive inhibitors for the hydrolysis of cerebroside-3-sulphate with an inhibition constant of 0.2 mM. 6. Kinetic parameters, metal ion effect and heat inactivation profile of enzyme suggest that the same active site of enzyme is responsible for the hydrolysis of cerebroside 3-sulphate, seminolipid and sulphogalactosylsphingosine.     

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

金黄色葡萄球菌对碘伏、乙醇、高铁酸钾和紫外线的抗性

目的了解临床分离的耐甲氧西林金黄色葡萄球菌（methicillin resistant Staphylococcus aureus,MR-SA）、甲氧西林敏感的金黄色葡萄球菌（methicillin sensitive Staphylococcus aureus,MSSA）及金黄色葡萄球菌CMCC（B）26003对碘伏、乙醇医院常用消毒剂及紫外线的抗性。方法以临床连续收集的67株金黄色葡萄球菌为实验菌株。测定碘伏、乙醇、高铁酸钾对耐甲氧西林金黄色葡萄球菌（MRSA）、甲氧西林敏感的金黄色葡萄球菌（MSSA）和金黄色葡萄球菌CMCC（B）26003作用1、2min后的最小抑菌浓度（MBC）及紫外线作用不同时间后的杀菌试验,观察其对碘伏、乙醇、高铁酸钾及紫外线抗性的变化趋势。结果碘伏对MRSA的MBC明显高于MSSA及CMCC（B）26003,而乙醇、高铁酸钾对3种菌的最小抑菌浓度均相近,3种菌对紫外线抗性相近。结论在正常使用浓度范围内碘伏、乙醇、高铁酸钾对金黄色葡萄菌中无论是MRSA、MSSA都有很好的灭菌作用,且乙醇作用时间越长,抑菌效果越显著。紫外线在作用75min以上时,3种菌全部不能存活。     

Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction

By means of affinity labelling with N,N-dimethyl-2-phenylaziridinium ion (DPA) two forms of acetylcholinesterase were synthesized that contained one or two molecules of the label covalently attached to the enzyme. The reaction of native and covalently modified acetylcholinesterases with n-alkane sulfonyl chlorides CnH2n + 1SO2Cl at n = 1 -4 was used to characterize the reactivity and properties of the enzymes. It was found that labelling of acetylcholinesterase with one molecule of DPA did not affect the enzyme's reactivity. Acetylcholinesterase containing two labels (the second one presumably located at the anionic centre of the enzyme) displayed enhanced and more specific reactivity towards alkane sulfonyl chlorides. It was found that the phenomenon of acceleration caused by affinity modification is analogous to the influence of n-tetraalkylammonium ions on the same reaction. Therefore, the mechanism of regulation of the properties of the esteric centre, caused by affinity labelling of the enzyme at the anionic centre, is the same as in the case of n-tetralkylammonium ions.     

Inactivation of phosphorylase b by potassium ferrate, a new reactive analogue of the phosphate group.

Rabbit muscle phosphorylase b reacts with the phosphate-like reagent potassium ferrate, K2FeO4, a potent oxidizing agent. The reaction results in inactivation of the enzyme and abolition of the ability of the enzyme to bind 5'-AMP. Activating and nonactivating nucleotides which bind at the 5'-AMP binding site such as 5'-AMP, 2'-AMP, 3'-AMP, and 5'-IMP substantially protect the enzyme from inactivation by ferrate. One to two residues of tyrosine and approximately 1 residue of cysteine are modified by ferrate under the conditions employed. Tyrosine is protected by 5-AMP, whereas cysteine is not. The tyrosine modification is suggested as the inactivating chemical reaction. The location of the inactivating reaction is suggested to be in or near the 5'-AMP binding site. The structural and chemical properties of ferrate ion are discussed and compared to those of phosphate. Ferrate ion may be a reagent useful for phosphate group binding site-directed modification of proteins.     

Physical, immunochemical, and functional properties of Acanthamoeba profilin

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.