Purification,characterization, and biological activities of broccolini lectin

Plant lectins have displayed a variety of biological activities. In this study, for the first time, a 27 kDa arabinose‐ and mannose‐specific lectin from Broccolini (Brassica oleracea Italica × Alboglabra), named as BL (Broccolini lectin), was purified by an activity‐driven protocol. Mass spectrometry analysis and database search indicated that no matches with any plant lectin were found, but BL contained some peptide fragments (QQQGQQGQQLQQVISR, QQGQQQGQQGQQLQQVISR and VCNIPQVSVCPF QK). BL exhibited hemagglutinating activity against chicken erythrocytes at 4 µg/mL. BL retained full hemagglutinating activity at pH 7–8 and temperature 30–40°C, and had an optimal activity in Ca²⁺ solution. Bioactivity assay revealed that BL exhibited dose‐dependent inhibition activity on 5 bacterial species with IC₅₀ values of 178.82–350.93 μg/mL. Notably, 5‐fold reduction in IC₅₀ values was observed on normal L‐O2 vs cancerous HepG‐2 cells (924.35 vs. 178.82 μg/mL). This suggests that BL should be promising in food and medicine. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:736–743, 2015     

Purification of fetal rat hepatocytes

Fetal rat hepatocytes of greater than 90% purity from 16 and 20 day fetuses were prepared by combining the use of sedimentation at unit gravity and discontinuous Percoll gradients. With slight modification the method could also be used to purify 13 day fetal rat hepatocytes. At each day examined the method produced several subpopulations of hepatocytes. Hepatocytes were identified by morphology and the presence of alpha-fetoprotein. Fetal hepatocytes purified in this way were able to attach to a substratum, and retained the ability to synthesize alpha-fetoprotein in short-term culture.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.     

Purification and characterization of rat angiotensinogen

1. Angiotensinogen (renin substrate) was purified from plasma of nephrectomized rats by a four step procedure using ammonium sulfate fractionation, chromatography on Blue Sepharose CL-6B and SP-Sephadex C-50, and gel filtration on Sephadex G-150. 2. The final preparation had a specific concentration of 9.3 microgram angiotensin I/mg (mean of six separate runs). The best preparation so far obtained contains 14.6 microgram angiotensin I/mg protein, which represents a purity of 62%. 3. By sodium dodecyl sulfate disc electrophoresis an apparent molecular weight of 56,400, and by isoelectric focusing an isoelectric point of 4.85 has been determined. These properties of rat angiotensinogen are similar to those reported for human angiotensinogen.     

Purification and characterization of a liver-specific antigen.

A liver-specific antigen (F-antigen) previously demonstrated in saline extracts of BALB/c mouse liver by double immunodiffusion was isolated and characterized. The antigen was found widely distributed among mammals but absent from avian and frog liver extracts. In immunoelectrophoresis it had an electrophoretic mobility similar to that of serum beta2-globulins, was relatively thermolabile, and was precipitated at 30 to 70% saturated ammonium sulfate concentrations. Evidence was presented that this antigen is a protein or a moiety closely associated with protein. Gel-filtration on Sephadex G-200 revealed liver-specific antigenicity in the second peak. Ion-exchange chromatography on DEAE-Sephadex A-50 revealed four peaks of which only the third one exhibited liver-specific antigenicity. This active peak contained 11 polypeptides on SDS polyacrylamide gel electrophoresis. After electrophoresis on acrylamide gel in the absence of SDS, antigenic activity was detected on one fast-moving band. Extraction of the protein band followed by SDS gel electrophoresis showed one major component of m.w. 75,000 and two major bands of m.w. 72,000 and 93,000, respectively.     

Purification and characterization of a human pancreas-specific antigen

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.     

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase

Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the V(max) was 930 micromol/h/mg and K(m) was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50 degree C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase.     

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.     

Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor

The mesoderm of Xenopus laevis is formed through an inductive interaction in which a signal from the vegetal hemisphere of the blastula acts on overlying animal pole cells. We have recently reported that the Xenopus XTC cell line secretes a mesoderm-inducing factor (MIF) which may resemble the natural signal. In this paper, we describe the purification and biological effects of XTC-MIF. XTC-MIF is a hydrophobic protein with an isoelectric point of 7.8 and an apparent relative molecular mass (Mr) of 23,500. On reduction, XTC-MIF loses its biological activity and the protein dissociates into two inactive subunits with apparent Mr of about 15,000. These properties closely resemble those of transforming growth factor type beta (TGF-beta), and it is interesting that TGF-beta 2 has recently been shown to have mesoderm-inducing activity. The biological response to XTC-MIF is graded. After exposure to 0.2-1.0 ng ml-1 XTC-MIF, stage-8 animal pole explants form mesenchyme and mesothelium. At higher concentrations, up to about 5 ng ml-1, muscle is formed, occasionally with neural tissue. In response to concentrations of XTC-MIF greater than 5-10 ng ml-1, notochord and neural tissue are usually formed. The formation of notochord and neural tissue in response to XTC-MIF represents a qualitative difference between this inducing factor and the other known group of MIFs, the heparin-binding growth factors.     

Purification and characterization of rat liver transglutaminase

1. Transglutaminase (EC 2.3.2.13) was purified from rat liver. 2. The enzyme was stable at 25 degrees C in the pH range of 6.0-9.0, with the optimum at pH 9.0. 3. The enzyme was inactivated after incubation for 20, 4 and 1 min at 44 degrees C, 52 degrees C, and 60 degrees C, respectively. 4. Activation energies were 30.4 kcal/mol for denaturation and 19.9 kcal/mol for substrate conversion to products. 5. The enzyme was inactivated by sulfhydryl modification with hydroxymercuribenzoate (99.1%) and N-ethylmalemide (78.5%). 6. Calcium, required for the activity, was replaced to a lesser extent, by Mg2+, Sr2+, Zn2+ and Mn2+ (31.8, 27.0, 24.6 and 3.5%). 7. Steady-state kinetics showed: Vmax = 10 microM-min-1, Km = 0.05 mM (N-dimethylated casein), kcat = 31.9 min-1 kcat/Km = 560 min-1 mM-1.     

Purification and characterization of rat liver glutaminase

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.     

Purification and characterization of rat urinary renin

Rat urinary renin was purified by a procedure involving ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B chromatography, ion exchange chromatography and gel filtration. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 39000 by SDS-gel electrophoresis and 40000 by gel filtration. The optimum pH determined with rat angiotensinogen was 7.0, and the Km was 3.6 microM. These properties agreed well with those of purified rat renal renin. The activity of urinary renin was specifically inhibited by anti-renin antibody. These results suggest that urinary renin may originate in the kidney.     

Purification and characterization of rat plasma kallikrein

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.     

Fliv-1: a surface membrane differentiation antigen of fetal rat hepatocytes

A monoclonal antibody of IgG1 subclass was raised against 15 day fetal hepatocytes. The antibody appears to recognize a surface membrane-associated fetal hepatocyte antigen. Preliminary experiments suggest that the antibody recognizes a protein epitope. The tissue distribution of the antigen, designated Fliv-1, was studied by immunoperoxidase staining on whole body sections from fetal rats of different gestational ages. In 15 day fetal rats the antigen was detected on Rathke's pouch, brain infundibulum, tongue, liver, pancreatic acini, adrenal gland, vertebral myotome, and some developing muscle tissue. All normal adult tissues that were examined, two rat hepatoma cell lines, and hepatomas induced in rats by a single injection of diethylnitrosamine, were negative for the Fliv-1 antigen. On the basis of its tissue distribution, it seems unlikely that Fliv-1 is a previously described fetal hepatocyte marker.     

Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.     

Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a

The production of halocins, bacteriocin-like proteins of ecological significance, is a frequent characteristic of species from the family Halobacteriaceae. Halocin H1, produced by Haloferax mediterranei strain M2a, is a single 31-kDa polypeptide. Its purification was achieved by combining two chromatographic systems: Sepharose 4B linked to bacitracin followed by hydroxylapatite Bio-gel HTP. Halocin H1 required concentrations of NaCl higher than 1.5 M to maintain its activity. Haoarchaeal strains showed a differential degree of sensitivity to the action of this halocin. Electronic Publication     

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.     

A1 adenosine receptor of rat testis membranes. Purification and partial characterization

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.     

Purification and characterization of gamma-glutamyltransferase from rat pancreas

gamma-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-gamma-glutamyl-p-nitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylglycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney gamma-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.