Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Purification and characterization of lysophospholipase D from rat brain

A lysophospholipase D (lysoPLD) was purified to apparent homogeneity from rat brain nuclear fractions using 1-[(14)C]palmitoyl-glycerophosphorylcholine as a substrate. The abundance of autotaxin (ATX), a secretory lysoPLD, was also estimated for each fraction. The nuclear fraction had relatively high levels of lysoPLD activity but weak immunoreactivity with an anti-ATX antibody. LysoPLD activity was further purified 5550-fold by sequential chromatography. The final preparation migrated as a single band with a molecular weight of 35,000. Anti-ATX antibodies did not cross-react with the purified enzyme. Moreover, enzyme activity was highest at pH 7.0-7.5 and requires Mg(2+). The Km and Vmax values for 1-palmitoyl-glycerophosphorylcholine were 176 microM and 0.3 micromol/min/mg, respectively. The purified enzyme hydrolyzed saturated forms of LPC more robustly than unsaturated forms. The enzyme could hydrolyze platelet-activating factor (PAF) to the same extent as 16:0-LPC, and showed a higher activity toward lysoPAF (1-O-hexadecyl-2-lyso-glycerophosphorylcholine). These results suggested that the lysoPLD purified from rat brain nuclear fractions in this work is a novel enzyme that hydrolyzes lysoPAF, PAF, and LPC to liberate choline.     

Purification and characterization of perchloric acid soluble protein from rat lung

We isolated a perchloric acid soluble protein from the post-mitochondria supernatant fraction of the rat lung and designated it as RLu-PSP1. The protein is soluble in 5% perchloric acid and was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The amino acid sequence of RLu-PSP was identical with that of rat liver PSP (RL-PSP). RLu-PSP inhibited protein synthesis in a rabbit reticulocyte lysate system. It was expressed mainly in cytoplasm of bronchioles and alveolar epithelial cells of the lung from 60-day-old rats. In 15-day-old rat embryos, the epithelial-lining of the terminal buds of the respiratory tree was immunopositive. The expression of RLu-PSP increased from the embryonic 15th day to the postnatal 40th day. This is the first report on the presence of a PSP in rat lung and on its involvement in the regulation of cellular growth and differentiation.     

Purification and characterization of two types of fumarase from Escherichia coli

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.     

Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes.

1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer Mr of each reductase was determined to be 32,000 +/- 1300. 3. Vmax values of reductase II for the various electron acceptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome b5 were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE-cellulose column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.

Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.     

Purification and characterization of enkephalinase B from rat brain membrane

Enkephalinase B from rat brain membrane which hydrolyzes enkephalin at the Gly-Gly bond was purified about 9400-fold to apparent electrophoretic homogeneity. The enzyme, which has a molecular weight of 82,000, consists of a single polypeptide chain. The enzyme has a pH optimum of 6.0-6.5 and is stable in the neutral pH region. The Km values of Met-enkephalin and Leu-enkephalin for this enzyme were 5.3 X 10(-5) M and 5.0 X 10(-5) M, respectively. The enzyme was inactivated by metal chelators, EDTA and o-phenanthroline and restored by the addition of divalent metal ions, Zn2+, Mn2+ or Fe2+, but was not inhibited by bestatin, amastatin, phosphoramidon or captopril. The enzyme hydrolyzed Met-enkephalin and Leu-enkephalin effectively. Although the enzyme belongs to the dipeptidyl aminopeptidase class, enkephalin-related peptides such as Leu-enkephalin-Arg, dynorphin (1-13) or alpha-endorphin and other biologically active peptides examined were hardly, or not at all, hydrolyzed. It was assumed that enkephalinase B functions mainly in enkephalin degradation in vivo.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Purification and characterization of palmitoyl-CoA ligase from rat brain microsomes

We have previously shown the existence of two separate enzymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA in rat brain microsomal membranes (1). Palmitoyl-CoA ligase activity was solubilized from brain microsomal membranes with 0.3% Triton X-100 and purified 93-fold by a combination of protein purification techniques. The Km values for the substrates palmitic acid, CoASH and ATP were 11.7 microM, 5.88 microM and 3.77 mM respectively. During activation of palmitic acid ATP is hydrolyzed to AMP and pyrophosphate, as evidenced by the inhibition of this activation by 5 mM concentrations of AMP, pyrophosphate or AMP and pyrophosphate to 70%, 60% and 85% respectively. The divalent metal ion Mg2+ was required for activity; its replacement with Mn2+ resulted in a 35% decrease in activity. Palmitoyl-CoA ligase activity was inhibited by the addition of oleic or stearic acids whereas addition of lignoceric acid or behenic acid had no effect. This supports our previous observation that palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes in rat brain microsomal membranes.     

Purification and partial characterization of rat liver soluble catechol-O-methyltransferase

The rat liver soluble catechol-O-methyltransferase (EC 2.1.1.6.) has been purified utilizing a combination of conventional chromatography and HPLC. The purified enzyme has a molecular mass of 25 kDa, a pI of 5.1, and exists in two forms which differ in the nature of their intramolecular disulfide bonds. This difference causes these two protein forms to behave differently in reversed phase chromatography.     

Purification and characterization of two distinct dipeptidyl aminopeptidases in soluble fraction from monkey brain and their action on enkephalins

Two distinct dipeptidyl aminopeptidases, which were designated DPP-A and DPP-B, were purified from soluble fraction of monkey brain using Leu-enkephalin as the substrate. The enzymes were purified 187 and 136 fold, respectively. Both enzymes showed the optimum pH in neutral range. Their molecular weights were almost equal and were estimated to be about 100,000. Their Km values with Leu-enkephalin as the substrate were 5.6 X 10(-5) and 1.1 X 10(-5) M, respectively. Among synthesized substrates, the highest affinity of the enzymes was toward arginyl-arginine beta-naphthylamide with the Km values of 6.25 X 10(-5) and 6.41 X 10(-5) M, respectively. Both enzyme activities were inhibited by the metal-chelators DFP and PCMB. Two hundred fifty microM arphamenine A inhibited DPP-A and -B with inhibition of 36.6% and 44.1%, respectively. Beta-endorphin, ACTH, and glucagon inhibited only DPP-B, while beta-lipotropin and angiotensin II inhibited both DPP-A and -B when Leu-enkephalin was used as the substrate.     

Purification and characterization of the inositol 1,4,5-trisphosphate receptor protein from rat vas deferens

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Purification and characterization of two soluble cytochromes from the alkalophile Bacillus firmus RAB

A soluble cytochrome c and soluble cytochrome b were purified from the alkalophilic Bacillus firmus RAB. The cytochrome c, with an alpha band at 552 nm, had an apparent molecular weight of 16,500 and was acidic, with a pI of 3.4. At both pH 7.0 and 8.3, the midpoint potential of c-552 was +66 mV. Above pH 8.3, the cytochrome exhibited a pH-dependent decrease in midpoint potential. This property, among others, distinguished the cytochrome c-552 from other membrane-associated c-type cytochromes. The soluble cytochrome b, with an alpha band maximum at 558 nm, had a molecular weight of approx. 15,500 and was also an acidic protein, with a pI of 3.07. It exhibited a pH-independent midpoint potential of +28 mV.     

Purification and characterization of a substance P-degrading endopeptidase from rat brain

A novel substance P-degrading endopeptidase has been solubilized with Brij 35 from a membrane fraction of rat brain and purified by a procedure involving DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-100 gel filtration, and Mono-Q HPLC. The activity of the degrading enzyme was monitored by measuring the disappearance of substance P by means of a bioassay and HPLC. SDS-polyacrylamide gel electrophoresis under reducing conditions of the enzyme gave a single band corresponding to a molecular weight of 58,000. The molecular weight of the enzyme was estimated to be 55,000 by gel filtration and the optimum pH for its activity was 7.5.. The purified enzyme cleaved substance P at three bonds, Pro4-Gln5, Gln5-Gln6, and Gln6-Phe7, in the ratio of 2:2:3. EDTA, o-phenanthroline, and p-chloromercuribenzenesulfonic acid strongly inhibited the enzyme, while diisopropyl fluorophosphate, E-64, Z-Gly-ProCH2Cl, phosphoramidon, and captopril had little or no inhibitory effect on it. The cleavage of substance P by the rat brain synaptic membrane was also analyzed under the conditions with or without these inhibitors. The inhibitor-susceptibility of the cleavage sites suggests that the present enzyme, together with endopeptidase-24.11, is involved in the degradation of substance P in the synaptic region.     

Purification,characterization and identification of tryptophan aminotransferase from rat brain

Tryptophan aminotransferase was purified from rat brain extracts. The purified enzyme had an isoelectric point at pH 6.2 and a pH optimum near 8.0. On electrophoresis the enzyme migrated to the anode. The enzyme was active with oxaloacetate or 2-oxoglutarate as amino acceptor but not with pyruvate, and utilized various L-amino acids as amino donors. With 2-oxoglutarate, the order of effectiveness of the L-amino acids was aspartate > 5-hydroxytryptophan > tryptophan > tyrosine > phenylalanine. Aminotransferase activity of the enzyme towards tryptophan was inhibited by L-glutamate. Sucrose density gradient centrifugation gave a molecular weight of approx. 55,000. The enzyme was present in both the cytosol and synaptosomal cytosol, but not in the mitochondria. The isoelectric focusing profile of tryptophan: oxaloacetate aminotransferase activity was identical with that of L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) activity, with both subcellular fractions. On the basis of these data, it is suggested that the enzyme is identical with the cytosol aspartate: 2-oxoglutarate aminotransferase.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.