Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Affinity chromatography of alpha-chymotrypsin, subtilism, and metalloendopeptidases on carbobenzoxy-L-phenylalanyl-triehtylenetetraminyl-sepharose.

Carbobenzoxy-L-phenylalanyl-triethylenetetraminyl-Sepharose (Z-L-Phe-T-Sepharose) was found to be an effective affinity adsorbent for bovine pancreatic alpha-chymotrypsin [EC 3.4.21.1] as well as neutral [EC 3.4.24.4] and alkaline [EC 3.4.21.14] proteases of Bacillus species. These enzymes were adsorbed in the neutral pH range. alpha-Chymotrypsin was recovered by elution with 0.1 A acetic acid while neutral subtilopeptidase was eluted with 0.5 M NaCl at pH 0. Thermolysin and subtilisin were found in eluates with 1.5 and 2.0 M guanidine-HCl at pH 7.2, respectively. The resulting enzymes appeared homogeneous on disc-electrophoresis and showed higher specific activities than those of crystalline or highly purified preparations available commercially. Modifications of the active site serines of alpha-chymotrypsin and subtilisin by treatment with diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF) resulted in loss in their binding abilities to the adsorbent. Complexes of porcine alpha2-macroglobulin with each of these four enzymes and that of Streptomyces-subtilisin inhibitor (S-SI) with subtilisin were also found in nonadsorbed fractions.     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).     

A new affinity adsorbent for guanyloribonuclease. Guanylyl-(2'-5')-guanosine coupled to aminohexyl-Sepharose.

Guanylyl-(2'-5')-guanosine binds to RNase T1 in 1:1 stoichiometry with a dissociation constant of 0.22 mM at pH 5.0 and 25 degrees C. This nucleotide, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3.1.4.8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N1 greater than RNase F1 greater than RNase T1 greater than RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T1 and the adsorbent is weakened by the presence of a low concentration of 2', 3'-, or 5'-GMP, which are competitive inhibitors of the enzyme. RNase F1 was purified to homogeneity by use of this affinity adsorbent.     

Affinity chromatographic purification of human lysozyme, with special reference to human leukemia lysozyme.

Lysozyme [EC 3.2.1.17] was purified from human tears, serum, and urine of acute monocytic leukemia patients, renal disease patients, and residents in cadmium-polluted areas of Tsushima Island using an affinity adsorbent containing lysozyme-lysate of Micrococcus lysodeikticus cell walls as the ligand. By means of this procedure, leukemia lysozyme was purified 100- to 200-fold with an activity recovery of 80%. It was crystallized at pH 10. This purified preparation appeared homogeneous in disc electrophoresis and showed a specific activity 2.5-fold higher than that of crystalline lysozyme from hen egg-white. Tear lysozyme was also purified to a nearly homogeneous state while the enzymes from normal serum and urine of a nephrosis patient and of residents in cadmium-polluted area were still disc electrophoretically heterogeneous and showed low specific activity as compared with purified leukemia lysozyme.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.     

Study of protein adsorption on indigo particles confirms the existence of enzyme--indigo interaction sites in cellulase molecules

Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57-111 and 0-62 microg x mg(-1), respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Characterization of rat and human steroid sulfatases

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.     

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.     

Effect of beta-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3-beta-glucanase, a purified beta-glucosidase, and of partially purified endo-1,3-beta-glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied. Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F1, an alpha-glucan; F3, a beta-glucan; F4, a chitin-glucan; and F4b, a beta-glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation. The enzymes were found to degrade fraction F4b (beta-glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

Enzymes with new biochemical properties in the pectinolytic complex produced by Aspergillus niger MIUG 16

A comprehensive study on the purification and characterization of pectinolytic enzymes produced by Aspergillus niger MIUG 16 on raw materials solid-state fermentation is reported. Five pectinolytic enzymes were purified using a combination of chromatographic techniques. The properties of these homogenous enzymes were analyzed. The purified enzymes were classified with respect to their biochemical properties and substrate specificity. Among these proteins, one revealed polygalacturonase activity, another appeared to be a pectin methylesterase and three were identified as pectate lyases. The capacity of the fungus A. niger to produce pectate lyases with optimum pH in acidic domain was reported for the first time.     

Isolation and characterization of dihydropteridine reductase from human liver.

Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.     

The substance reacting with SRBC (sheep red blood cells) and rabbit IgG. Isolation under mild conditions from rat thymus

A factor reacting with SRBC and rabbit IgG was obtained under mild conditions from rat thymus and spleen.The isolation procedure includes incubation of thymocytes or splenocytes with IgG-cellulose adsorbent, destruction of cells, washing the adsorbent and elution of an adsorbed material at pH 2. This preparation as well as the purified substance previously obtained by affinity chromatography on IgG-cellulose columns were found to agglutinate both SRBC and autologous erythrocytes. Preincubation in 1% SDS leads to dissociation of the preparation into several components separated by gel electrophoresis.A probable relation of this structure to the rosette forming capacity of T-lymphocytes is discussed.     

Pectic enzymes in pectolyase: separation, characterization, and induction of ethylene in fruits

The pectic enzymes in Pectolyase were separated by ion exchange chromatography on Q-Sepharose. Three pectin lyases, two polygalacturonases, and a pectinmethylesterase were resolved. The enzymes were further purified on Mono Q and/or Mono S columns to remove traces of cellulase. The enzymes had molecular weights ranging from 25,000 to 36,000 daltons. They were optimally active between pH 4.0 and 6.2 and were not greatly affected by ions. The pectin lyases and polygalacturonases were endo-enzymes. They solubilized uronic acids from washed cell wall fragments, but the lyases were much more effective than the polygalacturonases. The mixture of enzymes constituting Pectolyase increased ethylene production 15- to 25-fold when introduced into tomato and orange fruits. The enzymes purified from Pectolyase all increased ethylene production in the fruits but the lyases were generally more effective than the hydrolases.     

Differential salt-promoted chromatography for protein purification.

A range of hydrophobic-type adsorbents for protein chromatography has been screened for the binding, at high salt concentrations, of 10 enzymes from a bacterial extract. Adsorbents were chosen for tandem chromatography, in which the first adsorbent removed much of the protein, and the second and subsequent columns bound the desired enzymes. Simple schemes for isolating Zymomonas mobilis and yeast alcohol dehydrogenases are described, in which the enzymes are affinity eluted by NAD+.     

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.