Beta-cyanoalanine synthase: Its purification and basic physico-chemical properties]

A method has been developed for the purification of beta-cyano-L-alanine synthase from etiolated 10-day-old seedlings of blue lupine. High purity preparations of the enzyme were obtained with specific activity exceeding 4000-fold that of the seedling homogenate. Preparations were homogeneous on electrophoresis in polyacrylamide gel. The yield of total activity after purification was approximately 20%. Glutamic acid is the enzyme's only N-terminal amino acid; the molecular weight of the enzyme (both native and treated with 6 M urea) is 52000. The synthase containes one mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4,8. The enzyme's absorption spectrum has a maximum at 410 nm i.e., in the characteristic range of many pyridoxal-U-containing enzymes. Data on the amino acid composition of the enzyme are presented.     

Stability and catalytic properties of penicillin acylase in systems with low water content

Stability and catalytic properties of native and immobilized penicillin acylase were studied in systems with low water content. Preparations of both native and immobilized penicillin acylase demonstrated the catalytic activity even in solid-phase systems which contained 3-5 wt. % of water. The stability and catalytic activity of penicillin acylase at low water content depended on the thermodynamic water activity (aw) in the system.     

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.     

Aromatase cytochrome P-450. Purification and characterization of the enzyme from human placenta

Aromatase cytochrome P-450 (P-450arom) was purified from human placental microsomes. Preparations exhibit a single major band of approximately 55 kDa on SDS-polyacrylamide gel electrophoresis and have a specific content of 11-13 nmol P-450/mg protein. The purified enzyme exhibits spectral properties typical of ferric and ferrous forms of cytochromes P-450. Full enzymatic activity can be reconstituted with rabbit liver P-450 reductase, and catalytic characteristics similar to aromatase in microsomes are observed. Rabbit antibodies to purified P-450arom were affinity purified and show high specificity and sensitivity on immunoblots.     

Subunit hybridization and immunological studies of duplicated phosphoglucose isomerase isozymes

We examined structural and functional properties of the recently duplicated cytosolic isozymes of phosphoglucose isomerase (PGI) (EC 5.3.1.9) of the wild plant Clarkia xantiana and related species. A new purification protocol yielded PGIs with high specific activities. The duplicated PGI isozymes showed similar substrate affinities (apparent Km) in both catalytic directions. A newly devised competitive enzyme-linked immunosorbent assay (ELISA), using polyclonal antibodies, distinguished sodium dodecyl sulfate (SDS)-denatured PGIs coded by the duplicated loci but did not discriminate between allelic products of one of the loci. In vitro dissociation and reassociation experiments revealed that the duplicated subunits differed in their efficiency of reassociation. The difference was closely correlated with differences between the isozymes in their in vivo accumulation. In contrast, allelic subunits in species with or without the duplication were able to reassociate with similar efficiency. The duplicated PGI isozymes have diverged more in structural properties than kinetic ones. The total evidence suggests that mechanisms have evolved which reduce the potential consequences of the duplication.     

Separation of cyclic 3',5'-AMP from ATP, ADP, and 5'-AMP by precipitation with inorganic compounds

The antibiotic anisomycin is a very useful tool in studying protein synthesis since it is a specific inhibitor of the peptidyl transferase centre of eukaryotic ribosomes (5–7). By tritium exchange labeling followed by chromatographic and electrophoretic purification, we have obtained [³H]anisomycin of specific activity 285 mCi/mmole, and the methodology followed is described in this paper. This method is useful in preparing tritium labeled antibiotics other than anisomycin provided that the nonradioactive compound has the following characteristics: (a) a chemical structure resistant to the method required for tritium labeling, (b) ionic groups, and (c) chromophore groups with absorption maxima in the uv or visible part of the spectrum. Since these circumstances concur frequently in a number of chemical structures, a method essentially similar to that described in this work might be widely used. The method was not applicable to amicetin, blasticidin S, and fusidic acid, as these antibiotics were broken down during the tritium labeling. However, gougerotin, a well known inhibitor of peptide bond formation by prokaryotic and eukaryotic ribosomes (2–7), has been tritiated and purified following a method very similar to that described in this contribution to [³H]gougerotin (110 mCi/mmole) (16).     

Implication of pH in the catalytic properties of anthrax lethal factor

The anthrax lethal factor (LF) is a Zn(2+)-endopeptidase specific for mitogen-activated protein kinase kinases (MAPKKs), which are cleaved within their N-terminal region. Much line of effort was carried out to elucidate the catalytic activity of LF for designing the inhibitor and to understand the cellular mechanism of its cytotoxicity. Current assay methods to analyze the LF activity have been based on a synthetic peptide, consisting of 15-20 residues around being cleaved. However, there are accumulating reports that the region distal to cleavage site is required for the LF-mediated proteolysis of substrate. In this study, we demonstrate the catalytic properties of LF, using the full-length native substrate, MEK. We described the catalytic properties of LF focused on the effects of the pH alteration, which was encountered during the endocytosis of lethal toxin, and of the requirement for metal ions. We present the first evidence that additional metal ions are required for the LF catalyzed hydrolysis of native substrate, and that the pH alteration causes a significant change of catalytic properties of LF.     

Active centers of gamma-glutamyltransferase in the aerosol OT reverse micellar system in octane by an inhibitor analysis method]

Regulation of the supramolecular structure and catalytic activity of the heterodimeric enzyme gamma-glutamyltransferase in the system of Aerosol OT reversed micelles in octane was studied. Variation of the hydration degree causes a reversible dissociation of the enzyme to the light and heavy subunits, both possessing the catalytic activity. The subunits were separated on the preparative scale in the reversed micelle system using ultracentrifugation. The active centres of gamma-glutamyltransferase were studied using the enzyme's irreversible inhibitor AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). It is shown that the separation of the gamma-glutamyltransferase subunits results in "opening" of a new active centre in the heavy subunit, whereas in the enzyme's dimeric form this centre is masked and not accessible to the inhibitor's molecule. The kinetic and inhibitor analysis data indicate that the active centres in the light and heavy subunits are similar.     

Protein inhibitor of acid deoxyribonucleases. Improved purification procedure and properties.

A method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate versus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme.     

Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains. Demonstration of differential sensitivity to kinase inhibitors.

Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type 1 receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.     

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.     

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.     

Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties.

1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 mumol NADH formed min-1 mg protein-1. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60 000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30 000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km = 0.37 mM, galacturonic acid Km = 54 muM and NAD+ (with glucuronic acid) Km = 80 muM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 muM. 10. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site.     

Inhibitors of animal cell-free protein synthesis from grains

We described earlier the purification and properties of a protein (tritin) from wheat that enzymatically inhibits translation in cell-free systems from animals but not plants. In this report, we have examined 11 additional grains (Family Gramineae) and three other seeds for the presence of tritin-like proteins. In addition to wheat species, barley, oats, rye, triticale and corn were found to be sources of inhibitor; no inhibitor could be detected in rice, millet, sesame, alfalfa, mung bean or common bean seeds. The inhibitors from barley and rye were purified and found to differ from tritin with respect to heat inactivation, although they are similar to tritin with respect to molecular weight, behavior during purification and specific activity. The inhibitor from corn was purified and found to differ from tritin with respect to heat inactivation and molecular weight, although it is similar to tritin in behavior during purification and specific activity. These inhibitors constitute 2–17% of the total extractable protein in these grain s. Thus, wheat, barley, rye and corn can serve as convenient sources of a family of closely related inhibitors of protein synthesis which, when conjugated with lectins, antibodies, or hormones, could prove useful as chimeric toxins.     

Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium

A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.     

Purification of the Proteinaceous α-Amylase Inhibitor from Phaseolus Vulgaris by Affinity Chromatography with Immobilized Enzyme or Antibody

A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.     

Characterization and purification of a Ca2+ ion-activated neutral proteinase inhibitor in rabbit skeletal muscle

This paper describes the isolation, purification and properties of a specific inhibitor of calcium-activated neutral proteinase (CaANP) in rabbit skeletal muscle. The inhibitor was a thermo-acid-stable protein degraded by trypsin and chymotrypsin and seemed to contain two polypeptide chains with molecular weights of 70 000 and 13 000 daltons. Maximal inhibitory activity was obtained at neutral pH. High salt concentrations were needed to suppressinhibition. Inhibitor concentration had no effect on the optimal Ca++ ion levels for CaANP. These experiments also show that enzyme inhibitor association was instantaneous and did not need any incubation.