Isolation and properties of lipoproteins from normal rat serum

Three major classes of lipoproteins (VLDL, d <1.006; LDL, d 1.006-1.040; HDL, d 1.063-1.21) were isolated by ultracentrifugal flotation from the serum of normal male Sprague-Dawley rats. Their physical, chemical, and immunological properties were analyzed and compared with those of their water-soluble, essentially lipid-free derivatives. Studies were also carried out on the d > 1.21 fractions. Each product was found to have distinct characteristics, and this was also indicated by spectral analyses carried out by the techniques of circular dichroism and UV absorption spectroscopy. The results provided evidence for the mutual role of the protein and lipids in determining the structure, and perhaps the immunological specificity, of serum lipoproteins.     

Isolation and partial characterization of rat urinary esterase A2

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.     

Isolation and characterization of a fatty acyl esterase from rat lung

In an effort to facilitate studies of the reaction involved in the removal of fatty acids from acyl proteins, we have synthesized an octanoic acid ester of doubly blocked serine, specifically octanoyl N-carbobenzoxy-L-serine-benzyl ester (octanoyl boc-serine), and used it as a substrate to guide the purification of an esterase from rat lung. The esterase was purified 228-fold by column chromatography on DE-52 cellulose, hydroxylapatite, octyl-Sepharose, and concanavalin A-Sepharose and by HPLC gel filtration. The final enzyme preparation ran as a single 77,000-Da band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited a single symmetrical peak (sedimentation coefficient, 4.5 S) when centrifuged through a sucrose density gradient (empirical Mr, 63,000). The esterase is an acidic protein, pI 4.1, and is very active against p-nitrophenyl esters comprised of C4-C14 fatty acids; the highest specific activity (26.5 mumol/min/mg) was obtained using p-nitrophenyl caprylate as substrate. The pH optimum of the lung esterase is near 8.0 and the activity on octanoyl boc-serine is maximum when 0.3% (w/v) Myrj-52 is included in the assay medium. The activity of the esterase is not dependent on calcium ions. The enzyme does not remove acyl groups from the G-protein of vesicular stomatitis virus or the proteolipid of bovine brain. The possible role of the esterase in the metabolism of acylated proteins is considered.     

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the ¹⁴C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.     

Isolation of hexokinase II isoenzyme from the hyaloplasm of rat skeletal muscle

A method for obtaining partially purified preparation of II isozyme of hexokinase from cytosol of the rat skeletal muscles has been suggested. According to the data of electrophoretic and kinetic analyses the preparation does not practically contain I isozyme of hexokinase and is characterized by high enzymatic activity. The obtained preparation of II isozyme of hexokinase may be successfully used for research of the adsorption mechanism controlling the enzyme activity.     

酯酶同工酶在烟草远缘杂种早代中的应用

为了扩大种质资源的范围,将紫苏、罗勒的DNA运用常规杂交手段转入普通烟草中,从而获得新的烟草后代。通过研究烟草杂种早代酯酶同工酶的差异,结果表明:在子代中具有与父本相同的遗传物质,即父本部分遗传物质在烟草中的整合与表达。     

Purification and some properties of an esterase from yeast

An esterase was isolated and purified from baker's yeast by ammonium sulfate precipitation and column chromatographies on Sephacryl S-200, DEAE-Sephacel, chromatofocusing, and DEAE-Sephacel again. The molecular weight of the enzyme was approximately 84,000 on Sephadex G-100 and 40,000 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, suggesting a dimer for the activity. This enzyme hydrolyzed short-chain naphthyl esters and p-nitrophenyl esters, and its activity was strongly inhibited by mercuric compounds. The esterase appeared to be an arylesterase (EC 3.1.1.2) and its optimum pH was 8.0 at 30°C.     

Isolation and physico-chemical properties of rat ceruloplasmin

Ceruloplasmin was isolated and purified from albino rat blood serum. Relative molecular mass of the protein is 130 000. Electrophoresis of the protein preparations leads to a formation of the apo-protein devoid of the oxidase activity and migrating slower than the holo-protein. Leucine was found to be the N-terminal amino acid of the ceruloplasmin polypeptide chain. The amino acid composition and carbohydrate content of the protein were determined. The tryptic peptide maps of rat ceruloplasmin were compared to those of human protein. The properties of rat and human ceruloplasmin are discussed with respect to copper metabolism in animal body as well as in normal humans and patients with Wilson's disease.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Re-examination on the sex-influenced esterase (ESSI) in rat serum

The sex-limitation of sex-influenced esterase (ESSI) in serum of rats carrying Es-Sia allele was re-examined. ESSI was detected in immature males and females, and orchiectomized rats as well as mature females whereas ESSI in normal males rapidly disappeared with puberty. The rats orchiectomized at weaning temporarily lost ESSI around the age of sexual maturation, thereafter, ESSI reappeared. When orchiectomized rats were administered testosterone, synthesis of ESSI was suppressed as in normal adult males. Effect of ovariectomy was not recognized. The exceptional strain named SI 3 of which normal mature males have a significant level of ESSI has been established, although the level in the males is lower than that in the females of the same strain.     

益母草不同组分的抑螺效果及对钉螺酯酶同工酶的影响

通过水浸液灭螺实验,对益母草的根、茎、叶水浸液及益母草水苏碱水溶液的杀灭钉螺作用进行了初步研究.结果表明:各处理均有较好的灭螺效果,但毒效较氯硝柳胺略慢,益母草各部分灭螺效果的顺序为叶＞茎＞根;采用同工酶电泳技术检测了益母草水浸液处理钉螺时对钉螺酯酶同工酶的影响,处理1～2 d后样品的酶活高于对照组,处理3～4 d后的酶活则大大减弱,在这一过程中,有时出现新的酶带,有时又有酶带消失,酶带的变化主要在正极区,与正常的病理反应完全一致.     

菜粉蝶不同发育期酯酶同工酶的比较研究

利用垂直板型聚丙烯酰胺凝胶电泳法研究菜粉蝶Pieris rapae不同发育期的酯酶同工酶,并探讨在个体发育过程中的作用。     

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.