Purification of MINUS: A negative regulator of microtubule nucleation in a variety of organisms

Microtubules (MT) are important for cell behavior and maintenance, yet the factors regulating MT assembly in vivo remain obscure. In a biochemical search, we have isolated a small (4.7 kDa) acidic, phosphorylated polypeptide, which we named MINUS (microtubule nucleation suppressor) for its activity to inhibit MT nucleation [P. Fanara, B. Oback, K. Ashman, A. Podtelejnikov, R. Brandt, EMBO J. 18 (1999) 565]. Here, the purification strategy was optimized and the polypeptide purified to homogeneity from bovine brain, Drosophila, Caenorhabditis elegans and yeast. Amino acid analysis showed similar composition of MINUS from different species. In particular, MINUS was rich in glycine, threonine, isoleucine, leucine and acidic amino acids. Inductively coupled plasma mass spectrometry revealed a large peak for phosphorus confirming its identity as a phosphopeptide. For further purification, MINUS was separated as a single peak on reverse phase-HPLC (RP-HPLC). Preliminary sequence analysis suggested MINUS to be N-terminally blocked. However, conventional enzymatic digestions did not reveal differences in the peak profile compared to undigested MINUS. Hence, partial acid hydrolysis and proteinase K digestion was performed followed by RP-HPLC. The proteinase K digested peaks were subjected to Edman degradation (first peak, ser-pro-ser/gly-ser; second peak, tyr/arg-leu), mass spectrometry (no result) and MALDI analysis (no result). Collectively, the data suggest that MINUS belongs to a new class of MT assembly regulators. Sequence information and antibody development will be useful to examine its biological role in a definitive manner.     

Functional, genetic, and bioinformatic characterization of dextransucrase (DSRBCB4) gene in Leuconostoc mesenteroides B-1299CB4

A gene encoding a dextransucrase (dsrBCB4) that synthesizes only alpha-1,6-linked dextran was cloned from Leuconostoc mesenteroides B-1299CB4. The coding region consisted of an open reading frame (ORF) of 4,395 bp that coded a 1,465-amino-acids protein with a molecular mass 163,581 Da. The expressed recombinant DSRBCB4 (rDSRBCB4) synthesized oligosaccharides in the presence maltose or isomaltose as an acceptor, plus the products included alpha-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. Alignments of the amino acid sequence of DSRBCB4 with glucansucrases from Streptococcus and Leuconostoc identified conserved amino acid residues in the catalytic core that are critical for enzyme activity. The mutants D530N, E568Q, and D641N displayed a 98- to 10,000-fold reduction of total enzyme activity.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein.

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr. The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography. The purified protein is relatively heat stable (ca. 50% activity survives 30 min of boiling) and has a molecular weight of ca. 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis). It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye. Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E. coli HPr.     

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.     

Purification and properties of a histidine decarboxylase from Tetragenococcus muriaticus,a halophilic lactic acid bacterium

AIMS: A histidine decarboxylase from Tetragenococcus muriaticus, a halophilic histamine-producing bacterium isolated from Japanese fermented squid liver sauce, was purified to homogeneity, for the first time. METHODS AND RESULTS: The enzyme was purified 16-fold from cell-free extract by ammonium sulphate precipitation, anion exchange chromatography and hydroxyapatite chromatography. The pure enzyme consisted of two polypeptide chains with molecular mass of 28.8 and 13.4 kDa. The N-terminal amino acid sequences of these polypeptides highly correlated with those of the alpha- and beta-chains of other Gram-positive bacterial histidine decarboxylases. The optimum and stable pH for the enzyme was 4.5-7.0 and 4.0-7.0, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan and ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the Vmax and Km values were 16.8 micromol histamine min-1 mg-1 and 0.74 mmol l-1, respectively. CONCLUSIONS: The very similar physiological properties of this enzyme and almost identical N-terminal amino acid sequences to those from other Gram-positive bacteria indicated that this enzyme may be evolutionally highly conserved among Gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on this enzyme could be useful for studying the mechanism of histamine accumulation in salted foods. In addition, the N-terminal amino acid sequence can be utilized to design oligonucleotide probes, which may prove valuable in the rapid monitoring of halophilic histamine producers in salted products.     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp. paracasei CHCC 2115

AIM: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. METHODS AND RESULTS: The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.     

Human glucose 6-phosphate dehydrogenase: Purification and characterization of negro type variant (A+) and comparison with normal enzyme (B+)

Human glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.49) (A+), an electrophoretically distinguishable variant found in Negroes, was purified by column chromatographic techniques. The sedimentation patterns of analytical ultracentrifugation and interference patterns of sedimentation equilibrium indicate a homogeneous preparation. The molecular weight (by sedimentation equilibrium method) was estimated as 230,000, which was closely similar to that of the normal wild type enzyme (B+). The sedimentation constant of the variant enzyme (S _20,w=9.0) was smaller than that of the B+ enzyme (S _20,w=10.0). The molecular weight was about 45,000 in 4 mguanidine hydrochloride, indicating that the A+ enzyme, as well as the B+ enzyme, consisted of six subunits of similar size. The optimal pH of the variant enzyme was slightly higher than that of the B+ enzyme. In contrast to the B+ enzyme, magnesium ion increased the A+ enzyme activity with NAD as substrate. The Michaelis constants and the turnover rate were similar to those of the B+ enzyme. The A+ enzyme was serologically indistinguishable from the B+ enzyme when the anti-B+ serum was used as antibody. No significant difference was found in the amino acid composition of acid hydrolysates of the B+ and the A+ enzymes. This does not exclude an amino acid substitution, and, in fact, a single amino acid substitution, i.e., asparagine in B+ and aspartic acid in A+ enzyme, has been found and is being being reported separately.Supported by Research Grant HD-02497-01 and H-3901 from the National Institutes of Health.     

Purification and properties of rat brain hexokinase

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose-6-P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexokinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry6, 1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged by the amino acid composition and the relatively low molar extinction coefficient.     

Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase. Localized replacements in the carboxyl-terminal alpha-helix cause marked alterations in allosteric properties and intersubunit interactions.

A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax. In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km. Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.     

Purification and characterization of thiol aminopeptidase from the cytosolic fraction of human placenta

A thiol-dependent aminopeptidase was purified from the cytosolic fraction of human placenta. The purified enzyme consisted of a single polypeptide chain with a mol wt of 95,000. The enzyme was most active in the neutral region with Ala-pNA as substrate, and the activity was increased about 20-fold in the presence of some -SH compounds. The results of substrate specificity studies indicated that the enzyme hydrolyzes bonds involving the amino groups of neutral and basic amino acid residues. However, higher thiol-dependent activity was only detected with neutral ones. The enzyme was strongly inhibited by microbial aminopeptidase inhibitors, puromycin, o-phenanthroline, and sulfhydryl reactive-reagents. As to several naturally occurring peptides tested, the enzyme showed N-terminal Tyr-releasing activity toward enkephalins and kinin-converting activity.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Purification and N-terminal amino acid sequence of fructose-6-phosphate phosphoketolase from Bifidobacterium longum BB536

AIMS: The key enzyme in the fructose-6-phosphate shunt in bifidobacteria, Fructose-6-phosphate phosphoketolase (F6PPK; E.C. 4.1.2.22.), was purified to electrophoretic homogeneity for the first time from Bifidobacterium longum (BB536). METHODS AND RESULTS: A three-step procedure comprising acetone fractionation followed by fast protein liquid chromatography (FPLC) resulted in a 30-fold purification. The purified enzyme had a molecular mass of 300 +/- 5 kDa as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 93 +/- 1 kDa and 59 +/- 0.5 kDa, as determined by SDS-PAGE. CONCLUSION: The deduced N-terminal amino acid sequences of the two subunits revealed no significant similarity between them and other proteins when compared to the data bases of EMBL and SWISS-PROT, indicating that this could be the first report on N-terminal amino acid sequence of F6PPK. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study will be used to design oligonucleotide probe specific for bifidobacteria and to study the gene encoded F6PPK.     

Purification and characterization of a glutathione S-transferase from benoxacor-treated maize (Zea mays).

A glutathione S-transferase (GST) isozyme from maize (Zea mays Pioneer hybrid 3906) treated with the dichloroacetamide herbicide safener benoxacor (CGA-154281) was purified to homogeneity and partially characterized. The enzyme, assayed with metolachlor as a substrate, was purified approximately 200-fold by ammonium sulfate precipitation, anion-exchange chromatography on Mono Q resins, and affinity chromatography on S-hexylglutathione agarose from total GST activity present in etiolated shoots. The purified protein migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) as a single band with a molecular mass of 27 kD. Using nondenaturing PAGE, we determined that the native protein has a molecular mass of about 57 kD and that the protein exists as a dimer. Two-dimensional electrophoresis revealed only a single protein with an isoelectric point of 5.75 and molecular mass of 27 kD. These results further suggest that the protein exists as a homodimer of two identical 27-kD subunits. The enzyme was most active with substrates possessing a chloroacetamide structure. trans-Cinnamic acid and 1-chloro-2,4-dinitrobenzene were not effective substrates. Apparent Km values for the enzyme were 10.8 microM for the chloroacetamide metolachlor and 292 microM for glutathione. The enzyme was active from pH 6 to 9, with a pH optimum between 7.5 and 8. An apparently blocked amino terminus of the intact protein prevented direct amino acid sequencing. The enzyme was digested with trypsin, and the amino acid sequences of several peptide fragments were obtained. The sequence information for the isolated GST we have designated "GST IV" indicates that the enzyme is a unique maize GST but shares some homology with maize GSTs I and III.     

DNA-dependent protein methylase activity in bull seminal plasma.

The existence of a DNA-dependent protein methylase activity without any concomitant DNA methylase activity was demonstrated in bull seminal plasma. The enzyme utilized S-adenosyl-L-methionine as a methyl donor, and endogenous seminal plasma protein as the substrate. There was no demonstrable enzyme activity when the seminal plasma was preheated at 100 degrees for 10 min, or when the enzyme reaction mixture was incubated at 4 degrees. The protein methylase required a heterologous DNA source, had optimal activity at pH 8.1, and was enhanced in the presence of Mg2+, NH4+, and reduced glutathione. After the methylated protein product was separated from DNA by extraction with 0.2 M HCl, the incorporated radioactivity was shown to be totally solubilized by incubating the protein either with Pronase or 1 M NaOH, while RNase and DNase had no effect. Approximately 70% of the enzymatically synthesized amino acids in the protein product were tentatively identified as O-methylated amino acid ethers by virtue of their elution from a Dowex 50 H+ column with 0.2 M pyridine, and their stability to acid and base hydrolysis. The partially purified methylated product was shown by Sephadex G-50 chromatography to consist of three distinct radioactive proteins with molecular weights of approximately 21,000, 15,000, and 10,000.     

Characteristics of hepatic serine-pyruvate aminotransferase in different mammalian species.

1. Serine-pyruvate aminotransferase was purified from mouse, rat, dog and cat liver. Each enzyme preparation was homogeneous as judged by polyacrylamide-disc-gel electrophoresis in the presence of sodium dodecyl sulphate. However, isoelectric focusing resulted in the detection of two or more active forms from enzyme preparations from dog, cat and mouse. A single active form was obtained with the rat enzyme. All four enzyme preparations had similar pH optima and molecular weights. 2. Both mouse and rat preparations catalysed transamination between a number of L-amino acids (serine, leucine, asparagine, methionine, glutamine, ornithine, histidine, phenylalanine or tyrosine) and pyruvate. Effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine as amino donor. The reverse transamination activity, with hydroxypyruvate and alanine as subtrates, was lower than with serine and pyruvate for both species. Serine-pyruvate aminotransferase activities were inhibited by isonicotinic acid hydrazide. 3. In contrast, both dog and cat enzyme preparations were highly specific for serine as amino donor with pyruvate, and utilized pyruvate and glyoxylate as effective amino acceptors. A little activity was detected with phenylpyruvate. The reverse activity was higher than with serine and pyruvate for both species. Serine-pyruvate amino-transferase activities were not inhibited by isonicotinic acid hydrazide.     

A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation

A single nucleotide polymorphism (SNP) resulting in a substitution from Gln to His was found in exon 4 of the CYP2B6 gene in Japanese. The frequency of the variant allele was found to be 19.9%. The mutant- and the wild-type enzymes were expressed in Escherichia coli, and the effects of the single amino acid substitution on the catalytic activity were examined by investigating the kinetic profiles of 7-ethoxycoumarin O-deethylase activity. The wild-type enzyme showed typical Michaelis-Menten kinetics, while the mutant-type enzyme represented the sigmoidal kinetics with a higher V(max) value compared to that of the wild-type enzyme. Eadie-Hofstee plots further revealed an existence of allosteric effects for the reaction catalyzed by the variant. This is the first evidence demonstrating that only one amino acid substitution, Gln172His, caused by natural SNP enhances the catalytic activity of CYP by obtaining the character of homotropic cooperativity.