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.     

Tritiation of alpha-bungarotoxin with N-succinimidyl [2,3-3H]propionate. A useful reagent for labelling proteins.

Mono[3H]propionyl-alpha-bungarotoxin, prepared with N-succinimidyl [2,3-3H]propionate (sp. radioactivity 50 Ci/mmol) and purified to homogeneity by electrofocusing, retains its biological activity and stability. Rate constants for its binding to acetylcholine receptor were 4.4-fold lower than for unlabelled toxin; no dissociation was detectable. Analysis of enzymic digests of toxin showed 3H is located mainly of entirely in epsilon-propionyl-lysine.     

A Simple Colorimetric Method for the Determination of Lysozyme Activity

Nondialyzable model melanoidin prepared from glucose-glycine was separated into 6 components by pH gradient elution on a copper chelated Sepharose 6B column. The separated components were measured for copper chelating activity, UV-VIS spectrophotometry, and electrofocusing. The UV-VIS spectra of these components were found to be almost the same, but the melanoidin components with stronger affinity to the Cu column were found to be smaller in Kd.

Their electrofocusing profiles were similar, and the electrofocused bands were categorized into four major groups with pi 2.5–4.0. Major chelating activity of the melanoidin components was due to a band of pI 2.7 on the electrofocusing.     

Production and purification ofStaphylococcus aureus toxic-shock toxin

Staphylococcus aureus strain 5761, isolated from a patient with toxic-shock syndrome, was used for the production of toxic-shock toxin. The medium used contained 4% bio-Trypcase and 1% yeast extract adjusted to pH 7. Production of 50 μg of toxic-shock toxin/ml of culture supernatant was obtained. The purification method involves removal of the toxin from the culture supernatant with Biorex 70 resin and purification by isoelectric focusing, on 2% (pH 3–10) ampholine-sucrose gradients, and gel filtration on Sephadex G-100. Three antigenically similar entities were isolated after electrofocusing, with a major component at isoionic point pH 7.4. The purified toxin migrated as a homogeneous protein with a molecular weight of 23,700 when tested by gel electrophoresis. Specific antibodies to toxic-shock toxin in rabbits were obtained after one subcutaneous injection of 5 μg enterotoxin.     

Electrofocusing of heparin: fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides.

Twenty-one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular-weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N- and O-desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular-weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.     

Hindered migration of amino acids toward their isoelectric positions in gel electrofocusing, and facilitation of the migration at high ionic strength

Amino acids with a largepI -pK_p difference are known to be poor carrier ampholytes in electrofocusing, exhibiting isoelectric zones with poor conductivity across as many as 4 pH units. Accordingly, radioactive amino acids of this type, e.g., glycine, are found to be distributed over the entire pH gradient formed by Ampholine in electrofocusing gels, while radioactive amino acids like histidine or glutamic acid with small pI - pK_p differences form single peaks at or near their pI's. When poor carrier ampholyte amino acids are subjected to gel electrofocusing in 0.1 KCl, their distribution sharpens into single peaks, at or near the pI, indistinguishable from those of the good carrier ampholyte amino acids. At an intermediate stage of peak coalescence of the original broad distributions of poor carrier ampholyte amino acids, in 0.01 KCl, acidic and basic peaks of amino acid can be observed, possibly analogous to acidie and basic distributions previously observed with labeled Ampholine. The rate of peak coalescence of anionic amino acids seems higher than that of the cationic species. The mechanism by which high ionic strength facilitates the condensation of poor carrier ampholyte amino acids at their pI remains unknown. Possibly, the current within zones of poor carrier ampholyte amino acids is insufficient, or poor carrier ampholyte amino acids are not sufficiently charged, to allow for electrophoretic migration of the bulk of loaded amino acid to its isoelectric position, unless the current density is increased by electrofocusing at high ionic strength. Alternatively, 0.1 KCl may interfere with electrovalent interactions between amino acids and isoelectric carrier ampholyte zones, analogous to the action of urea in preventing the interaction between polyanions and carrier ampholytes.     

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.     

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.     

Isolation and characterization of the K6 type yeast killer protein

The optimum production of K6 type yeast killer protein by Kluyveromyces fragilis NCYC 587 occurred at pH 4.0-4.4 and at 22-24 degrees C in a killer-zone assay test. The K6 killer protein was concentrated by acetone precipitation of the culture supernatant and purified by native polyacrylamide rod gel electrophoresis. The protein migrated as a single band on discontinuous gradient SDS polyacrylamide gel electrophoresis and had a molecular weight of 42,313. The isoelectric point of the K6 type protein was determined at pH 5.97 by high voltage vertical polyacrylamide gel electrofocusing. Western blot analysis revealed that the K6 killer toxin was a nonglycosylated protein.     

Purification and Properties of α-Glucosidase and Glucoamylase from Lentinus edodes (Berk.) Sing.

An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.

Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase.     

Con A — Peroxidase method: an improved procedure for staining S-glycoproteins in cellulose-acetate electrofocusing in crucifers

Summary In the analysis of stigma glycoproteins by cellulose acetate electrofocusing in self-incompatible crucifers, the staining method of the glycoproteins, described in the earlier report, has been improved by using Con A — peroxidase reactions to obtain a permanent profile of band patterns which are visible under day-light conditions. Identifying S alleles by the corresponding S-glycoproteins can be facilitated by the present S-glycoprotein analysis.     

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.     

Effects of suppressor mutations on nonallelic glucose 6-phosphate dehydrogenase mutants of Neurospora crassa

Suppressor mutations have been isolated for bal and col-2, two slow-growing and nonallelic morphological mutants of Neurospora that carry defective G6PDs. Both suppressor mutations are located on linkage group I but are unlinked to the particular mutant that they suppress. The bal suppressor (su-B) increases the growth rate of bal and produces a more spreading morphology. su-B also decreases the G6P K _m of G6PD in bal;su-B double mutants. The col-2 suppressor (su-C) has similar positive effects on the morphology of col-2 and influences the electrofocusing pattern of the col-2 G6PD. su-C is an unusual type of suppressor mutation in that, when present in a wild-type background, it affects the electrofocusing pattern and kinetic properties of the normal enzyme. The nature of the su-C mutation, plus the complex genetic control of the Neurospora G6PD, is discussed.This work has been supported in part by the National Science Foundation (GB 21227), by the National Institutes of Health (GM 16224), and by a grant-in-aid from the Research Corporation.     

Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae

A new toxin of Enterobacter cloacae able to lyse erythrocytes and leukocytes was found. Purification of the toxin was performed by salt precipitation, gel filtration, ion exchange and HPLC in C₈ column. SDS-PAGE electrophoresis showed more than one bank corresponding to the leukotoxin able to form polymers and aggregate like some pore-forming cytotoxins (RTX). In culture supernatant the toxin showed 1 HU/ml (hemolytic unit) and 1.5 LU/ml (leukotoxic unit); after purification it reached 15 HU/ml and 20 LU/ml. The ratio between HU and percentage red cells affected the lytic capacity. E. cloacae toxin stimulated the oxidative metabolism of neutrophils, but over 50 μg toxin/ml the stimulus ceased as it was shown by NBT assay due to cell death. Chemiluminescence evidenced an increase in superoxide anion generation, but an excess of toxin interfered with this stimulus, as was previously observed in HlyA Escherichia coli toxin. Cross-reaction was found by immunoblotting with this HlyA. E. cloacae toxin presented higher amounts of proline, valine, aspartic and glutamic acids than HlyA. E. cloacae toxin was similar to HlyA in the prescence of a glycine-rich DNA sequence and in the observed effect of calcium on toxin activity. E. cloacae toxin did not cross-react by immunoblotting with hemolysin HmpA of Proteus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electrofocusing-induced alteration in the net charge of human macrophage-stimulating protein

Macrophage-stimulating protein is a mammalian serum protein, detected by its capacity to render mouse resident peritoneal macrophages responsive to chemoattractants. The purified protein was shown by column electrofocusing over a period of 48 h to have an isoelectric point of 7.0. In this study we determined the isoelectric point of the native molecule by electrofocusing human serum on a flat bed of prefocused ampholytes. Values in the range of 5.5–6.2 were obtained. A shift in the isoelectric point to 7.0–7.6, without significant alteration in the biological activity of the molecule, occurred when electrofocusing was prolonged to 48 h or was carried out in 6 m urea.     

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.     

Differential expression of defense‐related proteins of rice and sheath blight symptoms in response to active and inactive Rhizoctonia solani toxin

The effects of the phyotoxin from the fungal pathogen Rhizoctonia solani, causing sheath blight on the expression of defense‐related proteins of rice were investigated. The toxin inactivated by chemical treatment and by the toxin‐inactivating enzyme α‐glucosidase produced by Trichoderma viride was used in the study along with the active toxin. Toxin inactivated by T. viride α‐glucosidase and sodium periodate caused significantly less damage and electrolyte leakage to test plants. The active toxin and the pathogen induced chitinase and ß‐1,3‐glucanase synthesis in rice plants, while the inactivated toxin did not have any effect on the expression of these pathogenesis‐related proteins. The toxin was found to suppress the peroxidase activity 72 h after inoculation and the inactivated toxin restored the activity as that of untreated plants. There was no remarkable change in phenylalanine ammonia lyase activity in rice sheath treated with both the forms of the toxin.     

Selection of a <Emphasis Type="Italic">Clostridium perfringens</Emphasis> type D epsilon toxin producer via dot-blot test

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Purification and Characterization of UDP-N-Acetylgalactosamine: κ-Casein Polypeptide N-Acetylgalactosaminyltransferase from Mammary Gland of Lactating Cow

UDP-N-acetyl-d-galactosamine: κ-casein polypeptide N-acetylgalactosaminyltransferase was purified from a crude Golgi apparatus of lactating bovine mammary gland after solubilization with Triton X-100. Through chromatography on DEAE-Sephadex A-50, apomucin-Sepharose 4B, FPLC mono S, and Sephacryl S-200, and then electrofocusing, the enzyme was purified up to 7500-fold from the homogenate.

The molecular weight of the enzyme was estimated at 200,000 from gel filtration. The pI value of the enzyme was 6.4 on electrofocusing. The purified enzyme transferred GalNAc from UDP-GalNAc, not to the carbohydrate chains but to the polypeptide chains of the substrates, κ-casein and mucin. The enzyme required Mn²⁺, DTT, and Triton X-100 for maximal activity. The Km value for UDP-GalNAc was 16.2μm. Km values for K-subcomponents 1 and 7, and apomucin were 1.15, 5.10, and 0.192mg/ml, and V_max values were 254, 259, and 581 nmol/hr/mg, respectively. Thermal stability and the effects of pH, milk components, lectins, and nucleotides were examined.

α_s1-Casein strongly inhibited GalNAc transfer to κ-casein. The inhibitory effect of α_s1-casein was canceled by the addition of Ca²⁺, which causes casein micelle formation. This means that the glycosylation of κ-casein occurs after casein micelle formation triggered by the accumulation of Ca²⁺ in vivo.