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1.
Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.  相似文献   

2.
Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.  相似文献   

3.
Dienelactone hydrolase from Pseudomonas sp. strain B13.   总被引:6,自引:5,他引:1       下载免费PDF全文
Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13.  相似文献   

4.
The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated choline-containing lipids such as phosphatidylcholine, lysophatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl posphate-mannose. The partially purified mannosyltrasnferase exhibits a Km of 1.33 μM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient certrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.  相似文献   

5.
The enzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxy-14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media. A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.  相似文献   

6.
His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.  相似文献   

7.
A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).  相似文献   

8.
The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.  相似文献   

9.
Industrial concentrates from Aspergillus niger culture filtrates were fractionated by ion-exchange and adsorption chromatography. Several other types of hydrolases were completely removed. Eight partially purified components were obtained. Using specific activity as an estimate of purification, one aryl-β-glucosidase was purified 35-fold. Another component showed 147-fold purification using a viscosimetric assay with carboxymethylcellulose as substrate. The aryl-β-glucosidase was distinctly more thermolabile than the carboxymethylcellulase.  相似文献   

10.
Human renal renin. Complete purification and characterization   总被引:10,自引:0,他引:10  
Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.  相似文献   

11.
A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivum) is described. Electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 x x 10(-3) mumol/min per mg protein) was obtained after purification in 61 times. The molecular weight of the enzyme, determined by gel chromatography, was 143 +/- 2 kDa. The optimal conditions for the enzyme were 37 degrees and pH 8.0. Homogeneous preparation of the lipase exhibited high thermal stability: over 20% of original activity was retained after incubation of the preparation at high temperatures (60-90 degrees) for 1 h at pH 8.0.  相似文献   

12.
Preparations of human leukocyte interferon obtained by multi-stage purification procedure exhibited ribonuclease activity with the optimum at pH 7.0--7.5. The enzyme possessed the endonuclease action mechanism. Most substances studied for their effect on the RNA-ase activity in human interferon preparations showed many of them to act on the enzyme in the same way as on other ribonucleases. However, dithioerythritie, a reducing agent for disulfide bounds, activated the ribonuclease in the interferon preparation, as distinct from the pancreatic ribonuclease, which was inhibited by this preparation. Patterns of protein and RNA-ase distribution were obtained by electrophoresis in polyacrylamide gel.  相似文献   

13.
A five-step procedure is described for preparing highly purified aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC.2.6.1.1) from cell-freee enzyme extracts of Pediococcus cerevisiae. An overall purification of 130-fold was achieved. Some of P. cerevisiae aspartate aminotransferase properties were studied, i.s. pH optimum (7.8--8.0), optimum of temperature (37 degrees), Michaelis constans for 4 enzyme substrates and substrate specificity of enzyme. The enzyme is very thermolabile. During purification the enzyme was stabilizated by 2-oxoglutarate. The highly purified preparation was stored in the solution containing ammonium sulphate. The obtained aspartate aminotransferase preparation was free of alanine and aromatic amino acids aminotransferase activites and did not reveal malate dehydrogenase activity.  相似文献   

14.
Red beet ( Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3–14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for I h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO3. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg2+ and K+. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N;N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K+ Rb+ Na+ Cs+ Li+ and the kinetics of K+ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.  相似文献   

15.

Background

Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis.

Methods

Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine.

Results

Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state.

Conclusions

This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.  相似文献   

16.
Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5-0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7-10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20-26% of the liposome-entrapped enzyme was found in the liver of which 60-69% was in the lysosomal fraction. Spleen contained 3.6-5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed.  相似文献   

17.
Entrapment of enzyme in liposomes, biodegradable lipid vesicles, offers an intriguing strategy for the intracellular delivery of these macromolecules to the lysosomal apparatus for enzyme replacement endeavors in selected lysosomal storage diseases. Therefore, the in vivo tissue and subcellular fate and effect on the subcellular distribution of endogenous lysosomal hydrolases was determined following intravenous administration of β-glucuronidase entrapped in positively and negatively charged liposomes into C3H/HeJ β-glucuronidase-deficient mice. Enzyme entrapped in negatively charged liposomes was rapidly cleared from the circulation (t12 ≈ 4 min); maximal tissue recovery, 75% of dose, was detected in the liver at 1 h, was maintained for 48 h and then gradually declined to non-detectable levels by 8 days. A similar circulatory clearance and reciprocal hepatic uptake was observed for positively charged liposomes; however, the β-glucuronidase was retained in murine liver for 11 days. Significant activity, 15% of dose, was found in the kidneys up to 1 and 4 days post-injection of positively and negatively charged liposomes, respectively. No activity was recovered in neural or other visceral tissues except in spleen and lungs (?5% of dose). Exogenous β-glucuronidase activity administered in negatively charged liposomes was primarily localized in the lysosomally-enriched hepatic subcellular fraction, compared to the predominantly soluble localization of exogenous activity entrapped in positively charged liposomes. Administration of negatively charged liposomes caused no detectable change in the subcellular localization of several endogenous lysosomal hydrolase activities compared to their distribution in untreated mice. In contrast, a marked but temporary translocation of these hydrolase activities into the soluble fraction was observed following the administration of positively charged liposomes, identifying possible deleterious effects on cellular physiology.  相似文献   

18.
The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on Sephadex G-150 and CM-cellulose. Our results demonstrate the presence of several forms of poly(U) polymerase activities, some requiring exogenous RNA and others possessing their own endogenous primer RNA.  相似文献   

19.
Artemis is a member of the β-CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5′–3′ exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]6-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni–agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease.  相似文献   

20.
Cibacron blue T_3GA与溴化氰活化的Sepharose 4B偶联后,产生一种能有效地分离有机磷水解酶的吸附剂。用0.15mol/L MgCl_2溶液从黄杆菌P3—2细胞抽提出的粗酶液通过柱层析分离,即可得到纯化8倍、酶活性回收率为269.4%的纯酶制品。该酶制品用凝胶电泳测是均一的。  相似文献   

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