phenyl-Sepharose Chromatography Research Articles

Partial purification and characterization of chicken interleukin-2

Chicken interleukin 2 (IL-2) activity was partially purified from conditioned medium produced by culturing chicken splenic lymphocytes in the presence of concanavalin A. The purification procedure included sequential steps of gel filtration chromatography, reverse-phase high-pressure liquid chromatography, and phenyl-sepharose chromatography. Two peaks of IL-2 activity with apparent mol. wt. ranges of 36–39 kD and 17.5–25 kD were eluted from the Sephadex G100 gel filtration column. An increase in IL-2 spec. act. from 14 U mg −1 to between 2000 and 20 000 U mg −1 was obtained for the Sephadex G100 column peaks when subjected to the subsequent steps of the purification procedure. Alkylative reduction of the higher mol. wt. Sephadex G100 column peak (followed by re-chromatography with Sephadex G100), resulted in generation of the lower (17.5 kD) mol. wt. peak, indicating that chicken IL-2 is capable of either dimerizing or forming aggregates with other proteins. Elution of the lower mol. wt. IL-2 activity from a non-reducing sodium dodecyl sulfatepolyacrylamide gel demonstrated an apparent mol. wt. for chicken IL-2 of 20 kD, which confirmed the range of 17.5–25 kD seen with gel filtration.

Cloning and characterisation of members of a family of Babesia bigemina antigen genes containing repeated sequences

Bovine polyclonal antisera to Babesia bigemina antigens separated by phenyl-Sepharose chromatography were used to screen a B. bigemina λgt11 cDNA expression library. Eleven B. bigemina-specific cDNA clones were studied in detail. DNA sequencing of 2 representative clones identified open reading frames encoding polypeptides representing the carboxy-termini of 2 different proteins. Both polypeptides contained a related central motif of tandem repeats flanked by a highly conserved carboxy-terminal region, but the sequences preceding the repeats were not related. Hybridisation and restriction enzyme analysis of the cDNA clones indicated that they were derived from a family of at least nine related, but not identical genes. Four different members of the gene family have been isolated from a B. bigemina λEMBL3 genomic library. The genes are not closely linked and they occur on the largest and smallest B. bigemina chromosomes resolved by pulsed field gel electrophoresis (PFGE). Antibodies raised against the native antigens and purified on recombinant fusion proteins bound to multiple proteins (50–70 kDa) in the original B. bigemina antigenic fractions.

Enzymatic changes of the bovine pituitary multicatalytic proteinase complex, induced by magnesium ions

The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg 2+ markedly stimulated the breakdown of dephosphorylated β-casein (caseinolytic activity) and the hydrolysis of CbzLeuLeuGlu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of CbzD-AlaLeuArg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of CbzGlyGlyLeu p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg 2+ were measured at 52 °C rather than at 37 °C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated β-casein. Mg 2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 °C. Mg 2+ therefore converts a “repressed” form of MPC to an “activated” form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.

Purification and properties of NADP‐isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.

Purification and properties of factor XIII from human placenta

1. 1. FXIII was isolated and purified over 4000 fold from human placenta to apparent electrophoretic homogeneity by a new procedure inclucling ethanol precipitation. DEAE-Cellulose, molecular sieving on Sephacryl S-300 and Phenyl-Sepharose chromatography. 2. 2. Its pl was about 5.1. Under appropriate conditions, the incubation of FXIII in the presence of thrombin did not lead to inactivation cut in the polypeptidic chain. 3. 3. FXIII was also activated by CaCI 2 and, in a lesser extent, by other divalent cations like SrCl 2, Bad; or MgCl 2. 4. 4. The bincling of calcium to FXIII exhibited a negative cooperativity. 5. 5. The activity-pH curve of the calcium-activated enzyme did not appear very different from that of the thrombin-activated enzyme.

Human eosinophil cytotoxicity-enhancing factor: purification, physical characteristics, and partial amino acid sequence of an active polypeptide

Medium conditioned by PMA/LPS-stimulated U937 cells was processed for the purification of an eosinophil cytotoxicity-enhancing factor (ECEF) by the following sequence: 1) phenyl-Sepharose chromatography; 2) DEAE-cartridge chromatography; 3) preparative SDS-gel electrophoresis; and 4) reversed-phase HPLC. This resulted in the isolation of a 10 kDa polypeptide with ECEF activity. Purified material from 21 different preparations enhanced eosinophil killing of antibody-coated Schistosoma mansoni schistosomula by a mean of 206% (increase from 13.2 +/- 7.9% to 40.4 +/- 20.2% of targets killed, p less than 0.0001). Activity was maximal at a concentration of 20 ng ECEF polypeptide/ml and half-maximal between 0.8 and 4 ng/ml. Antibody specific for the 10 kDa polypeptide precipitated ECEF activity from a crude preparation and, by Western blot analysis, reacted only with a 10 kDa species in that preparation. The following N-terminal amino acid sequence was determined for the purified polypeptide: Val-Lys-Gln-Ile-Glu-Ser-Lys-Thr-Ala-Phe-Gln-Lys-Ala-Leu- -Ala- Gly- -Lys-Leu.... Computer search showed that this sequence is unrelated to other known protein sequences. Thus, the ECEF polypeptide is a newly defined monokine, with the ability to enhance eosinophil cytotoxic function in vitro. This monokine may be an important regulator of eosinophil function in inflammation in vivo.

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.

Expression of recombinant human apolipoprotein A-I in Chinese hamster ovary cells and Escherichia coli

Human recombinant apolipoprotein (apo) A-I was produced by Chinese hamster ovary (CHO) cells and Escherichia coli with expression vectors containing cDNAs encoding preproapoA-I or apoA-I, respectively. The apoA-I from CHO cells was purified from the culture medium by ammonium sulfate precipitation, phenyl-Sepharose chromatography, and affinity purification on anti-apoA-I immunoabsorber. Human apoA-I was produced in E. coli as a fusion protein with glutathione S-transferase. A four amino acid linker, which separated the two proteins, was specifically recognized and cut by Factor Xa. The purification was accomplished by chromatography of E. coli extracts on glutathione-Sepharose and digestion with Factor Xa. The highest production level was found to be 0.5 μg/ml of culture medium per 48 h for a clone of stable transformant of CHO cells, whereas E. coli could produce as much as 20 μg/ml of bacterial culture. These apoA-I forms were compared in terms of molecular weight, isoelectric point, and expression of several epitopes. Recombinant apoA-I obtained from CHO cells appears intact and its isoelectric point is compatible with that of the mature form and the proform of apoA-I, whereas a part of the apoA-I produced by E. coli does not contain the COOH-terminus. Also, two of six epitopes are expressed to a greater extent in apoA-I obtained from E. coli than in apoA-I obtained from human plasma.

Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system

The resolution and reconstitution of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system into a supernatant and a pellet fraction was accomplished by a procedure involving salt treatment followed by osmotic shock. Recombination of pellet and supernatant fractions was required for cyclase activity. This restoration effect could be demonstrated using either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester as the cyclase substrate in the presence or absence of S-adenosylmethionine. Pretreatment of the pellet fraction with either 8-hydroxyquinoline or desferal mesylate inhibited cyclase activity, indicating that there is a heavy-metal-ion requirement in this fraction. The cyclase supernatant protein(s) was not internalized by Sephadex G-50 and did not bind to Blue Sepharose, suggesting that it has a molecular mass of over 30 kDa and that it does not bind the cofactor NADPH. The cyclase supernatant protein did bind to MgProtoMe2-bound Sepharose and could be eluted by raising the pH to 9.7 in the presence of 4 mM-n-octyl glucoside. The pH optimum of the cyclase was 9.0. About a 40-fold purification of the cyclase supernatant protein was achieved by a combination of (NH4)2SO4 fractionation and phenyl-Sepharose chromatography.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.

Tissue Specific Expression of the Plasma Glutathione Peroxidase Gene in Rat Kidney1

Rat plasma glutathione peroxidase (GSH-Px) was purified 1,400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1,529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5 kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.

Covalent binding of dolichyl phosphate to proteins in rat liver

Rats were injected via the portal vein with (RS)-[5-3H]-mevalonolactone and the lipids were extracted. From fractions of liver homogenate, all labeled dolichol, cholesterol and ubiquinone could be extracted, but about 40% of microsomal and lysosomal dolichyl phosphate was only released after alkaline hydrolysis. Only a small amount of the non-extractable radioactivity was found to be associated with alpha-unsaturated polyprenyl phosphate. There was no difference in the polyisoprenoid pattern when the two pools of dolichyl phosphate were compared. On the other hand, the specific activity of the bound lipid was only half that of the extractable form. After phenyl-Sepharose chromatography, a peak of protein was isolated exhibiting a 25-fold enrichment in bound radioactive dolichyl phosphate. Treatment with a non-specific protease, followed by chromatography, gave polypeptide fragments associated with bound lipids. On SDS/PAGE a major protein band at 23 kDa and some minor bands with higher molecular masses were found to be associated with this lipid. The results indicate the presence of covalently bound dolichyl phosphate in rat liver.

Activation of Human Platelet Phospholipase C by Calpain-I

Effects of calpain-I purified from human erythrocytes on phosphatidylinositol hydrolysis activity of phospholipase C (PI-PLC) were investigated by using human platelet lysate and the partial purified enzymes. PI-PLC activity in human platelet lysate prepared in the absence of thiolprotease inhibitors (leupeptin and EST) was higher than that prepared in the presence of inhibitors. This result implied that PI-PLC was activated by thiolprotease in platelets during preparation of platelet lysate. When the thiolprotease inhibitors were removed by gel filtration from platelet lysate prepared in the presence of the inhibitors and calpain-I was added into the preparation, the PI-PLC activity was enhanced in correlation with the amount of calpain-I. PI-PLC in platelets lysate was partially purified by phenyl-Sepharose and Ultrogel AcA22 column chromatography. PI-PLC was separated to two isozymes by phenyl-Sepharose chromatography. The apparent molecular weight of these isozymes was about 200kDa in AcA22 gel filtrations. One of the PI-PLC, which was adsorbed with phenyl-Sepharose column and eluted with the buffer containing 50% ethyleneglycol, was not influenced by the preincubation with calpain-I, but the activity of the other one, which was not adsorbed with phenyl-Sepharose column, was enhanced three times by the preincubation with calpain-I. These results indicate that two isozymes of PI-PLC are located in platelets and one of the enzymes is activated by calpain-I.

The Erythrina Protease Inhibitor: Interactions with Tissue Plasminogen Activator

The Kunitz-type trypsin and tissue plasminogen activator (t-PA)-inhibitor from Erythrina caffra seeds was cleaved by trypsin at low pH to yield a disulphide linked two-chain molecule with reduced hydrophobicity. This change was used to separate cleaved from native inhibitor by phenyl-Sepharose chromatography. The inhibitor was not cleaved by t-PA. Trypsin, but not t-PA, catalysed resynthesis of the cleaved bond. Although the cleaved protein retained inhibitory activity for both trypsin and t-PA, 6-10 times higher concentrations were required for equivalent inhibition. Removal of the active site arginine (Arg63) from the cleaved inhibitor by digestion with carboxypeptidase B resulted in a further loss of inhibitory activity towards both proteases. The activity of the inhibitor could also be decreased by modification of one susceptible arginine residue with peptidyl arginine deiminase. These results suggest that the trypsin-reactive site of the Erythrina inhibitor is also involved in the interaction between the inhibitor and t-PA.

Strawberry Polyphenoloxidase: Purification and Characterization

ABSTRACTStable and highly active polyphenoloxidase (PPO) extracts were obtained using polyvinylpyrrolidone, Amberlite XAD‐4, Triton X‐100, and protease inhibitor in pH 5.25 buffer. Citrate and phosphate prevented binding of PPO to pectin. Phenyl sepharose chromatography resolved PPO into two isozymes (PPO‐F1 and PPO‐F2) both separated from pectin. PPO‐F1 had a molecular weight of 111,000, did not penetrate the running gel during electrophoresis, and was bound by concanavalin suggesting that it contained carbohydrate. PPO‐F2 resolved into two peaks during DEAE‐chromatography, had a molecular weight of 34,500, and resolved into two bands during electrophoresis.

Characterization and expression of the unique calmodulin gene of Aspergillus nidulans.

Complete cDNA and genomic clones for the unique calmodulin (CaM) gene of the filamentous fungus Aspergillus nidulans have been isolated and characterized. The gene contains five introns, of which three are at unique positions relative to other CaM genes. The A. nidulans CaM gene is transcribed as a single, 0.85-kilobase mRNA species that encodes a predicted protein 84% identical (93% similar if conservative changes are considered) to vertebrate CaM. The complete cDNA was ligated into a lambda PL promoter-regulated bacterial expression vector to allow expression of A. nidulans CaM in Escherichia coli. The expressed protein was purified from bacterial lysates by phenyl-Sepharose chromatography and migrated as a single species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Ca2+, A. nidulans CaM exhibited a shift in apparent Mr identical to vertebrate CaM. The bacterially synthesized protein activated vertebrate CaM-dependent phosphodiesterase, CaM-dependent protein kinase II, and myosin light chain kinase with kinetics similar to vertebrate CaM. Isolated conidia (G0 spores) were germinated to induce synchronous cell cycle re-entry and the levels of CaM mRNA and protein determined. Both CaM and its mRNA were regulated during cell cycle re-entry. Calmodulin mRNA levels increased 20-fold as germlings progressed through the G1 phase, while CaM levels increased 2-fold prior to the initiation of DNA synthesis. Messenger RNA levels decreased during S-phase while protein levels increased an additional 2-fold, peaking at the onset of mitosis followed by a subsequent decrease as cells completed mitosis. Disruption of the CaM gene by site-specific homologous recombination was lethal, indicating that CaM is essential for cell cycle progression.

Development of an Immunoassay for Human Serum Osteoclastic Tartrate-Resistant Acid Phosphatase*

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget's disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.

Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B).

Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55 degrees C. EDTA inhibits the activity, and this can be reactivated with Zn, Mn, and partially with Co. The reducing agents dithiothreitol and beta-mercaptoethanol and the divalent cation Cu inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K(m) of 0.15 mM and a V(max) of 151 mumol/min per mg of protein.

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40 degrees C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu and Cd. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or beta-mercapto-ethanol, while Zn or Co restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K(m), 0.55 mM) but that it can hydrolyze this substrate at a high rate (V(max), 30 mumol/min per mg of protein).