Fractionation of Nondialyzable Melanoidin into Components by Electrofocusing Electrophoresis

Nondialyzable melanoidin formed in a model system of glucose and glycine was applied on thin layer gel electrofocusing in a polyacrylamide gel. The electrofocusing profile differed according to the reaction time: the pI of the melanoidin formed in the early stage of the reaction was less than 2.9 and, as the reaction progressed, the pI of the melanoidin formed gradually shifted to a less acidic value, from 2.9 to 3.3. Those from the xylose and glycine model system gave a similar profile to that of the glucose system.

Preparative separation of the nondialyzable melanoidin, which was formed by heating the glucose system for 7hr, was performed by flat-bed electrofocusing in Sephadex gel. At least 14 melanoidin bands were clearly electrofocused at a pH range of 2.7-3.3 and about 59% of the melanoidin applied remained unaffected by electrophoresis at the starting position. The major component of electrofocused melanoidin was pI 3.00, this being made up of 12.5% of the total amount of electrofocused melanoidin. The molecular weight of melanoidin affected by electrophoresis was about 25,000, regardless of the pI value of the melanoidin components. The reducing activity, estimated by the potassium ferricyanide method, showed that the lower the pI of melanoidin, the higher was the reducing activity.

The addition of hydrochloric acid to the melanoidin solution caused it to gradually become viscous and the melanoidin was precipitated below pH 3.0-3.5, corresponding approximately to ampholite. This feature can be used as a method to prepare nondialyzable melanoidin in a short time.     

Screening of Microorganisms to Decolorize a Model Melanoidin and the Chemical Properties of a Microbially Treated Melanoidin

A microbe of Streptomyces werraensis TT 14, which was newly isolated from soil, decolorized the model melanoidin prepared from glucose and glycine, the decolorization rate being 64% in the optimal medium of pH 5.5 (2.0% starch, 1.0% yeast, 0.3% NaCl, and 0.3% CaC0₃) and 45% in a synthetic medium. There was virtually no difference in the UV-VIS absorption spectra of the microbially treated melanoidin and the control. The peaks of the gel permeation chromatogram for the treated melanoidin and for the control showed the same retention times, but lower molecular weight compounds increased in the decolorized melanoidin. The Cu(II)-chelating activity of the decolorized melanoidin was reduced to about half that of the control. The melanoidin component of pI 2.5 was increased and that of pI 3.5 was reduced by the microbial treatment.     

Isolation of Deoxyfructosazine and Its 6-Isomer from the Nondialyzable Melanoidin Hydrolyzate

The nondialyzable melanoidin prepared from glucose-ammonia system (kept in pH 5.3~6.0 during the reaction) was hydrolyzed. The hydrolyzate was fractionated by DEAE-cellulose column and Dowex 50 W column. Deoxyfructosazine and its 6-isomer were respectively isolated from main two fractions, and identified. Even on boiling the melanoidin in aqueous solution, these pyrazines as well as imidazoles and β-hydroxy pyridines in the melanoidin were liberated.

Furthermore, amounts of these heterocyclic compounds liberated from the nondialyzable melanoidin and the fractionated melanoidins (fractionated into five fractions on DEAE-cellulose column according to the method described previously) were examined.

The results obtained seem to suggest that these heterocyclic compounds are not present probably as a molecular skelton or an inclusion compound in the melanoidin, but as a small moiety of the melanoidin molecule with loose chemical bond.     

Relationship between the Molecular Weight and the Color Intensity of Color Components of Melanoidin from the Glycine-Xylose System

The molecular weights of color components (designated as P1, P2, P3, P4, P5, P6, P7 and P8 in order of elution from a DEAE-cellulose column) isolated by the conversion of color components of melanoidin produced from the glycine-xylose system in an oxidative browning were studied in relation to the color intensity. The molecular weights of P5, P6, P7 and P8 estimated by gel filtration on Sephadex G–50, G–75 and G–150 using dextran as a standard were approximately 2,140, 3,550, 5,600 and 14,200, respectively. The molecular weights of P1, P2, P3 and P4 could not be estimated by the gel filtration method because of their low values.

On the other hand, a linear relationship between Kd, the distribution coefficient in dextran gel, and E₄₅₀ (E) of the color components was observed. Thus, there is considered to be a linear relationship between log E and log molecular weight (M). The correlation coefficient between log E and log M was calculated to be from 0.96 to 0.98 in the visible wavelength region. Therefore, the equation, E=k × M^α was adopted. The value, E=2.15 × M^0.29 was obtained from melanoidin prepared from the glycine-xylose system. The molecular weights of P1, P2, P3 and P4 were calculated from the equation to be 290, 360, 700 and 1,200, respectively. The equation, E=k × M^α, was demonstrated to be reasonably applicable to the melanoidin from Glu-, Lys-, Gly₂-, Gly-Leu-, and Gly₃-xylose systems. It is concluded that the polymerization of the structural unit in melanoidin occurs in an oxidative browning and that their color tone is darkened and the color in melanoidin is increased by polymerization according to the equation, E=k × M^α.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Biochemical Properties of a Cephalosporin β-Lactamase from Pseudomonas aeruginosa

The cephalosporin β-lactamase from Pseudomonas aeruginosa GN918 was purified using CM-Sepha-dex column chromatography. The resulting preparation gave a single protein peak on electrofocusing column chromatography and a single protein band on polyacrylamide-gel electrophoresis. The specific enzyme activity was 22 950 units per mg of the purified enzyme protein. The optimal pH was 7.5 and the optimal temperature was 40 C for the hydrolysis of cephaloridine. Isoelectric point was 8.7 and the approximate molecular weight of the enzyme was found to be 34 000±2000. The enzyme activity was inhibited by iodine, p-chloromercuribenzoate and semi-synthetic penicillins. The enzymological properties of the isolated preparation have been compared with β-lactamases derived from other gram-negative enteric bacteria.     

Multiple forms of acidic endopeptidase from germinated barley

An endopeptidase preparation from germinated barley Hordeum vulgare L., cv. Trophy, purified by affinity chromatography and density-gradient electrofocusing, consisted of three or four components. The preparation was only partly resolved by electrofocusing, with evidence of three possible components (pI 4.15, 4.28, and 4.37). Gel filtration on Sephadex G-75 yielded an asymmetrical peak, the major part of which corresponded to a molecular weight of 14,100, with evidence of one larger and two smaller components. The activity of the preparation was sulfhydryl-dependent; cysteine was the most effective of several sulfhydryl compounds tested. The preparation was sensitive to O₂ in the absence of metal chelating agents and was inhibited by sulfhydryl reagents. It showed very narrow concentration tolerances for both cysteine and a substrate, N,N-dimethylhemoglobin. The Km value on N,N-dimethylhemoglobin at pH 3.8 was 0.064 to 0.067% (w/v) substrate; V_max was 0.80 to 0.83 A₃₄₀ per hour. Normal enzyme activity and molecular-size distribution were observed when the endopeptidases were extracted in the inhibited state and subsequently reactivated, thus ruling out the possibility that the enzymes might be autolytic artifacts that arose during extraction and purification.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Decolorization of Molasses Waste Water by a Thermophilic Strain,Aspergillus fumigatus G-2-6 Sadahiro

We screened for fungi that can decolorize molasses melanoidin in the tropical zone and isolated some strains, mainly in the genus Aspergillus. Of these, strain No. G-2–6 was most active and was identical with Aspergillus fumigatus based on detailed morphological studies.

This strain decolorized about 75% of a molasses melanoidin solution when the strain was cultivated on a glycerol-peptone medium at 45°C for 3 days with shaking. In successive decolorization reusing the mycelia, this strain had more than 60% of the melanoidin-decolorizing activity at the eighth replacement in the presence of 4% glycerol.

Continuous decolorization of molasses melanoidin solution in a jar fermentor had an almost constant decolorization yield of about 70% at a dilution rate of 0.014 hr^-1. At the same time, about 51 % of the chemical oxygen demand and 56% of the total organic carbon in the initial solution were removed. In contrast, continuous decolorization of non-dialyzed molasses melanoidin solution removed a little more chemical oxygen demand and total organic carbon than those of dialyzed molasses melanoidin solution, but had a lower level of melanoidin-decolorizing activity (about 40%).     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Penicillin Acylase of Pseudomonas melanogenum KY 3987

Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Anti-free-radical properties of the peptide fractions isolated from string bean by immobilized metal ion affinity chromatography

In this study, peptides and proteins extracted from string bean with 1% TCA were separated by chromatography on columns with copper ions immobilized through IDA and o-phosphoserine OPS. Protein and peptide concentrations and the anti-free-radical properties of the isolated fractions were determined. Identification was obtained using mass spectrometry. The anti-free- radical activity of all analyzed samples was determined using the DPPH test, and was found to depend on reaction time, choice of chelating agent and the order in which the fractions were eluted from the column.     

Purification and Some Properties of Melanoidin Decolorizing Enzymes,P-III and P-IV,from Mycelia of Coriolus versicolor Ps4a

Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48,400 ~ 50,000, an optimum pH of 5.5 and an optimum temperature of 30~35°C. P-III required glucose and 02 for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.

On the other hand, P-IV had a molecular weight of 43,800 ~ 45,000, an optimum pH of 4.0~4.5 and an optimum temperature of 30~35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CMB and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.

Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.     

Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (<1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml⁻¹ that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.     

Synthesis of Aspartyl Tripeptide Esters in Relation to Structural Features of Sweet Peptides

The distribution of multiple forms of β-amylase in some varieties or species of soybean seeds was examined by the gel isoelectric focusing method. Seven components (1′, 1, 2, 3, 4, 5 and 6) were found. Their respective isoelectric points were 5.07, 5.15, 5.25, 5.40, 5.55, 5.70 and 5.93±0.04. The varieties or species of soybean seeds were separated into two types by their zymograph: the low pI type and high pI type. Component 6 was purified from commercial defatted soybean meal containing all seven components by ion-exchange column chromatography and by gel filtration, and compared with previously purified components 2 and 4. Components 2, 4 and 6 had the same molecular weight and immunological properties but some differences were found in their amino acid compositions and enzymatic properties. The C-terminal amino acid of components 2 and 6 was glycine but that of component 4 was alanine. It was concluded from these results that differences between components 2, 4 and 6 were caused by charged amino acid substitution.     

Purification and Properties of Acid Carboxypeptidase I from Aspergillus oryzae

To elucidate the constitution of peptidases from Aspergillus oryzae, systematic separation of the enzymes was carried out by batchwise treatment with Amberlite IRC-50 and precipitation with rivanol. Proteases were separated to two fractions. They were Amberlite IRC-50 adsorbed and the non-adsorbed fractions and the latter fraction was further separated to two fractions, rivanol precipitable and non-precipitable fractions.

Acid carboxypeptidase I was purified from the rivanol non-precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and SE-cellulose. The purified enzyme was not homogeneous on disc electrophoresis, although symmetric peaks were obtained for enzyme protein and activity in Sephadex gel filtration. The optimum pH is at pH 4.0 for carbobenzoxy-l-alanyl-l-glutamic acid. The enzyme activity was inhibited by SH reagents, but not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 120,000 by gel filtration.     

Functional significance of the copper and lipid components of human ferroxidase-II.

Copper, phospholipids, and cholesterol remain tightly bound to the ferroxidase-II protein from human serum following extensive purification. In vivo studies with copper-deficient rats and in vitro studies with general and copper-specific chelating agents strongly suggest that the copper atoms associated with purified ferroxidase-II are extremely tightly bound and are essential for its catalytic activity. Only partial removal of the protein bound copper atoms can be achieved by treatment with chelating agents; however, virtually complete loss of the bound copper atoms accompanies the hydrolysis and removal of the bound lipid components. No dissociation or denaturation of ferroxidase-II occurs upon hydrolysis or removal of the bound lipid components. These results suggest that intact lipid components are necessary for the binding of copper to ferroxidase-II and that the association of the protein, lipid, and copper components is indispensable for the catalytic activity of ferroxidase-II.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Murine liver arylsulfatase B processing influenced by region on chromosome 17

SM/J liver arylsulfatase B has a more rapid electrophoretic mobility and occurs as a series of more acidic isozymes following electrofocusing in narrow pH gradients than the liver enzyme from C57BL/6J mice. The SM/J and C57BL/6J electrofocusing patterns were both converted to a single isozyme with similar isoelectric points by pretreatment with neuraminidase, suggesting that the SM/J and C57BL/6J isozymes differed with respect to their sialic acid content. Arylsulfatase B electrofocusing and thermostability phenotypes segregated independently among progeny of SM/J×C57BL/6J crosses, suggesting that the electrofocusing phenotypes were not determined by different alleles at As-1, the putative structural locus for arylsulfatase B. Comparison of the joint segregation of hepatic acid phosphatase electrophoretic patterns and liver arylsulfatase B electrofocusing profiles revealed that the electrofocusing profiles may be determined by a region on chromosome 17 near or identical to Apl. Kidney, brain, and spleen arylsulfatase B electrofocusing patterns did not appear to differ between SM/J and C57BL/6J mice.This research was supported in part by Biomedical Sciences Research Support Grant RR-07030, by NIGMS Grant 1-RO1GM27707-01, and by Grant 1–570 from the National Foundation/March of Dimes.