Identification,properties, and genetic control of UDP-glucose: Cyanidin-3-rhamnosyl-(1→6)-glucoside-5-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 5-hydroxyl group of cyanidin-3-rhamnosyl-(1→6)-glucoside has been demonstrated in petal extracts of Silene dioica plants. This glucosyltransferase activity was not detectable in green parts of these plants. The enzyme activity is controlled by a single dominant gene M; no glucosyltransferase activity could be demonstrated in petals of m/m plants. The enzyme was purified eightyfold by PVP and Sephadex G50 chromatography. The glucosyltransferase had a pH optimum of 7.4, had a molecular weight of about 55,000, was stimulated by divalent metal ions, and had a “true K_m” value of 0.5×10^?3 m for UDP-glucose and 3.6×10^?3 m for cyanidin-3-rhamnosylglucoside. Pelargonidin-3-rhamnosylglucoside also could serve as acceptor. The enzyme did not catalyze the glucosylation of the 5-hydroxyl group of cyanidin-3-glucoside, although in petals of M/- n/n mutants cyanidin-3,5-diglucoside is present. ADP-glucose could not serve as a glucosyl donor.     

Genetic control and biosynthesis of two new flavone-glycosides in the petals of Melandrium album

In a chemicogenetic analysis of the geographical distribution of flavone-glycosides in the petals of Melandrium album, we found two unknown flavone-glycosides in ten Hungarian and four German populations. By means of classical techniques for the identification and structure determination of flavonoids, the structure of these flavones turned out to be 6-C-glucosylglucosylapigenin and 7-O-glucosyl-6-C-glucosylglucosylapigenin, respectively. Genetic analysis showed that the coupling of glucose to the carbon-carbon bound glucose of isovitexin (6-C-glucosylapigenin) was controlled by a single dominant gene, Fg. Fg controls a UDP-glucose: isovitexin 6-C-glucosylglucosyltransferase. By means of ammonium sulfate fractionation and Sephadex chromatography, the enzyme was purified sixfold. The partly purified enzyme had a pH optimum between 8.0 and 8.5. The apparent K _m value for UDP-glucose in the presence of 1.0 mm isovitexin was 2.2 mm. The apparent K _m value for isovitexin in the presence of 1.8 mm UDP-glucose was 0.08 mm. The glucosyltransferase activity was stimulated by the divalent cations Mn, Mg, Co, and Ca. Neither 7-O-glucosyl nor 7-O-xylosyl isovitexin could serve as an enzyme substrate. Therefore, the biosynthesis of 7-O-glucosyl-6-C-glucosylglucosylapigenin found in the petals of M. album proceeds in a sequential manner: first the formation of 6-C-glucosylglucosylapigenin, followed by 7-O-glucosylation. Isovitexin 6-C-glucosylglucosyltransferase activity controlled by gene Fg could also be demonstrated in leaves of Fg plants. The enzyme probably uses another substrate in these green parts of the plant, because both isovitexin and isovitexin-glycosides are absent.     

The problem of isolation of the X virus inhibitor from potato leaf sap

Using moving boundary electrophoresis in a veronal buffer, pH 8·5, of 0·05 ionic strength and at 4·3 mA, we succeeded in separating the inhibitor, which was obtained by fractionation on a Sephadex G-50 column, into four components. The two most substantial components represent 25% and 65% respectively from the separated proteins as a whole. The heterogeneity of the inhibitor was proved by analytical ultracentrifugation, too. The ionex chromatography was applied for the quantitative separation of the inhibitor. We used ionex chromatography on DEAE Cellulose the concentration gradient being 0–1m NaCl in a 0·01m phosphate buffer, pH 7·3, and on DEAE Sephadex A-50 using the same concentration gradient in a phosphate buffer, of 0·1 ionic strength and pH 7·3. In both cases four components were obtained. The most substantial component, representing 65% of the whole analysed material, was eluted at the concentration 0·15–0·3m NaCl, and was electrophoretically homogenous and showed the most effective inhibitory ability.     

Enzyme preparations fromAspergillus flavus for structural studies of the peach gum polysaccharide

Extra- and intracellular glycanohydrolases were isolated fromAspergillus flavus and partially characterized. Both preparations exhibited β-galactosidase activity. Gel chromatography of the extracellular enzyme preparation on Sephadex revealed one protein fraction containing β-galactosidase activity and a second one exhibiting mainly β-xylosidase activity. Electrophoresis in starch gel and disc electrophoresis in polyacrylamide gel showed that the preparation obtained from the cultivation broth contained five protein fractions, whereas two protein fractions could be detected in the intracellular preparation. Hydrolysis of a partially degraded polysaccharide of peach gum by the above preparations yieldedd-galactose as the main product and traces ofd-mannose,l-arabinose,d-xylose and a number of oligosaccharides.     

The isolation of potato virus S from the leaves of potato plants using precipitation by polyethylene glycol

A purified potato virus S was prepared using precipitation by the solution of 35% polyethylene glycol 4000 in the presence of an electrolyte. The mixture for precipitation of the potato virus S had to contain 11% polyethylene glycol and 0·25m NaCl. The S virus was extracted by 0·01m phosphate buffer, pH 7·5, from the precipitation separated by centrifugation. One part of the extract was further purified by means of differential centrifugation and the other by means of gel filtration on Sephadex G-100. The ultraviolet absorption measurements of both preparations showed that the differential centrifugation gave a purer preparation than the gel filtration.     

Studies on choline acetyltransferase isolated from human brain

The purified choline acetyltransferase from human striatal tissue was found to have aK _m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH₄)₂SO₄, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.     

Solid-phase synthesis of ragweed allergen Ra5

A two-stage strategy was used to synthesize the 45-residue, four-disulfide-bonded ragweed protein allergen Ra5: (i) the protected linear chain was prepared by solid-phase, stepwise synthesis according to the sequence of the fully reduced protein; (ii) after removal from the resin and deprotection by hydrogen fluoride, the reduced chain was purified by gel filtration on Sephadex G-50 and allowed to fold “spontaneously” in the presence of oxidized dithiothreitol to form the disulfide links. The product (synthetic Ra5) was purified to disc-electrophoretic homogeneity by cycling through Sephadex G-50 and CM-Sephadex (overall yield: 6% theoretical). Synthetic Ra5 was closely similar in structure to the natural protein by amino acid analysis. polyacrylamide gel electrophoresis, tryptic peptide mapping, and circular dichroism spectra, as well asin vitro andin vivo immunoassays of antigenic/allergenic activities.     

Investigation of the properties of alcohol dehydrogenase from bean,rape, wheat and broad bean

Preparations of alcohol dehydrogenase (ADH) were prepared from germinating seeds by a procedure including fractionation with ammonium sulphate, desalting on a column of Sephadex G-25 and chromatography on DEAE-cellulose, the specific activity of ADH was in the case ofPhaseolus 31 times,Brassica 43 times,Triticum 47 times, andVicia 212 times higher than that of crude extract. The enzymes were homogeneous when filtrated on Sephadex G-200. Molecular weight of all the four studied ADH was approximately 63 000. Some kinetic properties as K_m for ethanol as substrate, substrate specificity towards different alcohols, and the effect of some intermediates of sugar metabolism and of some inhibitors on the activity of the enzymes were also followed. The results obtained are discussed with respect to possible mechanism of action of the plant ADH.     

Snake venom phosphodiesterase: A zinc metalloenzyme

Snake venom phosphodiesterase fromCrotalus adamanteus can be purified by blue sepharose chromatography followed by gel exclusion chromatography on Sephadex G-100. The enzyme is judged to be homogeneous by SDS gel electrophoresis. Atomic absorption spectrometry indicates a stoichiometry of 1.07 g-atom of zinc per mole of purified enzyme. The enzyme is inhibited by a wide variety of structurally different metal binding agents, e.g., 1,10-phenanthroline, thioglycolic acid,l-cysteine, 8-hydroxy-5-quinoline sulfonic acid, EDTA, and dipicolinic acid. The results of both the chelator inhibition and the atomic absorption analysis indicate that snake venom phosphodiesterase is a zinc metalloenzyme. Snake venom phosphodiesterase shares a number of mechanistic features in common with the nucleotidyl transferases. All of these enzymes contain zinc, are activated by magnesium, and catalyze α-β phosphoryl bond cleavage. Mechanistic studies of phosphodiesterase may therefore be helpful in understanding the mechanism of the hydrolytic step catalyzed by all of these enzymes.     

Purification and amino acid analysis of cytochrome c fromUstilago violacea

Cytochrome c fromUstilago violacea was further analyzed in order to characterize the pink phenotype. NaOH-extracted cytochrome c was purified in three steps, which included ammonium sulfate precipitation, CM-Sephadex ion-exchange chromatography with 0.5 N NaCl elution, and CM-Sephadex ion-exchange chromatography with a 0.0–0.6 N NaCl gradient elution. Polyacrylamide gel electrophoresis of the purified protein yielded a single red band, which was the only band detected upon Coumassie brilliant blue staining. HCl-hydrolysates of the protein were examined for their amino acid composition, which indicated that the cytochrome c fromU. violacea contains approximately 104 residues, with high levels of alanine, histidine, serine, and low levels of phenylalanine and arginine.     

Purification and properties of acetate kinase from Clostridium thermoaceticum

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min^-1 mg^-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K _m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S_0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S_20w value was 6.0 S.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^?) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^?) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

Isolation and purification ofβ-galactosidase ofLactobacillus murinus CNRZ 313

β-Galactosidase has been isolated inLactobacillus murinus CNRZ 313, and its properties have been studied. The enzyme was purified 292-fold by chromatography on Ultrogel ACA 34, DEAE-Sephadex A-50 columns, and by affinity chromatography in agarose-p-aminophenyl-β-d-thiogalactoside. The purified extract exhibited a single band following polyacrylamide gel electrophoresis (PAGE). Molecular weight was estimated to be 170,000 on an Ultrogel ACA 34 column. Maximum enzymatic activity was observed at 45°C and pH 7 in 50 mM phosphate buffer. The Km for ONPG and ONPG + 20 mM of lactose were 480 μM and 870 μM, respectively. The effect of different salts on the enzymatic activity was studied. An inhibitory effect was observed when using 10 mM CaCl₂, and a stimulating effect when using 10 mM MgCl₂. The latter protected the enzyme from thermal denaturation. Among the protective agents belonging to the sulfhydryl group that was tested, mercaptoethanol and dithiotreitol acted as activators, while glutathione and cysteine acted as inhibitors, of the enzymatic activity.p-β-Galactosidase activity was not observed.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.     

Growth distribution and surface pH patterns along maize roots

The distribution of elongation and surface pH patterns along the primary roots of maize (cv. LG 11), maintained vertically in humid air (darkness, 22°C), have been analysed quantitatively. A new technique employing Sephadex G 25 beads containing a pH indicator dye (bromocresol purple), was used for measuring both the growth gradient of the roots (Sephadex beads as markers) and at the same time, the surface pH changes (referring to a standard scale). The optimal axial growth was located between 2 and 4 mm from the tip. This coincides with the optimal decrease in surface pH.     

Relationship between the composition of the regulatory complex of esterases and the age ofMycobacterium phlei culture

During submerged cultivation ofMycobacterium phlei a mixture of macromolecular compounds ia released into the medium. Concentrated filtrates of cultures of different ages were separated on Sephadex G-25 fine and polyanions were found to predominate in the young culture. During further days of fermentation the proportion of polycations significantly increases. The results are discussed with respect to the regulatory complex of esterases.     

Evidence from two-protonic-state reactivity probe kinetics that chymopapain in fresh nonfruit latex ofCarica papaya consists of multiple forms of chymopapain A. The value of catalytic site characteristics in the identification,classification, and characterization of the papaya cysteine proteinases papain,the chymopapains,and papaya proteinase Ω

Fresh latex ofCarica papaya was collected from the stem, leaves, and petioles of the growing plant and fractionated by ion-exchange chromatography on a column of SP-Sephadex-C50 and by FPLC using a Mono S column. The fractions were examined for catalytic activity using Z-Lys-ONp andl-BAPNA as substrates and the thiol contents and reactivity characteristics were determined by using 2,2′-dipyridyl disulfide as a two-protonic-state thiol titrant and reactivity probe. By these methods the fresh nonfruit latex was shown to contain papain (EC 3.4.22.2), multiple forms of chymopapain, all of which have catalytic site reactivities characteristic of chymopapain A, and papaya proteinase Ω (originally called papaya peptidase A). The necessity now to characterize the catalytic site of a chymopapain in order to identify it is discussed.     

Purification and properties of a fructose-1,6-diphosphate activated l-lactate dehydrogenase from Staphylococcus epidermidis

l-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 yielded molecular weights of about 130000 and 126000 respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

Biochemical characterization of a metallothionein-like protein in rat brain

Recent investigations from this laboratory have identified a metallothionein-like protein in the rat brain with an elution volume (ve/vo) of 2.08 and a molecular weight of smaller than 10,000. The synthesis of this protein was stimulated following intracerebroventricular (icv, 0.20 μmol zinc/μL/h, 48 h), but not intraperitoneal (ip) administration of ZnSO₄. Furthermore, chronic ip administration of ZnSO₄ (5.0 mg/kg/d/10 d) did not alter the level of the metallothionein-like protein in the brain. However, the hepatic metallothionein was induced following icv administration of ZnSO₄. The chromatofocusing of metallothionein-like protein isolated by gel permeation chromatography on Sephadex G-75 exhibits three zinc-binding peaks, which focus on pH 6.8, 6.2, and 5.3, respectively. It is expected that the protein peak focusing at 5.3 is a metallothionein-like protein. Purification of the zinc stimulated metallothionein-like protein on ion exchange chromatography on DEAE-Sephadex A-25 columns, using a linear gradient elution procedure produced two isoforms, eluting, respectively, at 75 and 137 mM of Tris-acetate buffer, pH 7.5. The comparative high-performance liquid chromatographic (HPLC) profiles of the zinc-induced hepatic metallothionein isoforms I and II (retention times 17.39 and 18.73 min) and that of the zinc-stimulated metallothionein-like protein isoforms I and II (retention times 17.32 and 18.64 min) are very similar. The function(s) of the metallothionein-like protein isoforms in the brain remains to be elucidated.     

Purification and characterization of aβ-galactosidase fromTreponema phagedenis (Reiter strain)

A β-galactosidase was highly purified from a cellular extract ofTreponema phagedenis (Reiter strain) by ammonium sulfate precipitation and successive chromatography on Sepharose 6B and DEAE-Sephadex. The purified enzyme was homogeneous in SDS-polyacrylamide gel electrophoresis. The molecular weight estimated was 580,000. The optimal pH, ionic strength, and temperature were 6.5, 0.1, and 50°C, respecitvely. The enzyme was stable only at around pH 6.5 and at temperatures lower than 35°C. The enzyme was irreversibly inhibited byp-chloromercuribenzoate and divalent cations such as Hg²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺. The Km values forp-nitrophenyl-β-D-galactopyranoside,o-nitrophenyl-β-D-galactopyranoside, and lactose were 0.29, 0.36, and 5.4 mM, respectively.