A new method for the detection and assay of α-1,3-glucanases

A colorimetric method that is specific for the assay of α-1,3-glucanases is presented. The enzyme substrate consists of Cibacron Blue F3GA complexed with a dextranase-treated streptococcal glucan. The method is especially convenient for tests involving large numbers of samples, and can be adapted to quantitative as well as qualitative applications. The assay is sufficiently sensitive for screening bacterial samples as potential sources of α-1,3-glucanase.     

Optical properties of Cu(II) complexes with heparin and related glycosaminoglycans.

Absorption and circular dichroism measurements have been carried out to obtain information regarding the stability and the nature of complexes between Cu(II) and heparin, and between Cu(II) and related glycosaminoglycans. In the presence of Cu(II), all glycosaminoglycans, except keratan sulfate, show a characteristic absorption band near 237 nm, which we assign to charge-transfer bands involving ligands to metal ion. From the absorbance values, the formation constants of Cu(II)-heparin and Cu(II)-(itN)-desulfated heparin have been determined to be approximately 1 × 10⁴ and 2 × 10² mol^?1, respectively. The large difference in the stability constant values is attributed to the difference in the charge density of the polymers, and to involvement of more than one ligand in the case of heparin. The CD characteristics of the Cu(II)-heparin complex suggest that both carboxyl and sulfamino groups are involved as ligands. The appearance of extrinsic CD bands in heparin, heparan sulfate, dermatan sulfate, and N-desulfated heparin at pH > 5 is ascribed to asymmetry of chelate rings. Absence of CD change in chondroitin sulfate and N-desulfated heparin (pH < 5) in the presence of Cu(II) suggests that only the carboxyl group is involved in those complexes. The differences either in iduronic acid conformation (C-1 vs. 1-C) or in intersaccharide linkages between dermatan sulfate and heparin (or heparan sulfate) are revealed in the difference CD spectra between the complexes and the polymers. The change in the intrinsic Cotton effect on complex formation is accounted for as a change in spatial orientation of the ligand groups rather than as a major conformational change of the polymers.     

An assay for iduronate sulfatase (Hunter corrective factor)

Acetylation of benzyl α-D-mannopyranoside with acetic anhydride-sodium acetate at room temperature gave crystalline benzyl 2,3,6-tri-O-acetyl-α-D-manno-pyranoside (25%) and benzyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (≈65%). Similar esterification of benzyl β-D-glucopyranoside yielded the crystalline benzyl 2,4,6-triacetate (66%), whereas the corresponding galactopyranoside gave the crystalline 3,4,6-, 2,3,6-, and 2,4,6-triacetates (3, 25, and 9%. respectively). The structures of these compounds were established by methylation with diazomethane-boron trifluoride etherate and were confirmed by n.m.r. studies.     

A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

alpha-Galactosidase (melibiase) from Saccharomyces carlsbergensis: structrual and kinetic properties.

This report describes structural and kinetic properties of the purified α-galactosidase from Saccharomyces carlsbergensis. This galactosidase has many similar properties to other exocellular enzymes in yeast which have been reported. Its molecular weight of 300,000 is comparable; it has similar carbohydrate content (57%) and amino acid and carbohydrate composition. That is, 35% of its amino acid residues can be accounted for by threonine, serine, and aspartic acid. Its carbohydrate composition is primarily mannose (90–95%) with approximately 7% glucose and 1% glucosamine. The enzyme is very stable to both acidic and alkaline conditions as well as to heating to 50 °C. α-Galactosidase remains active after incubation with as much as 1% sodium dodecyl sulfate at 30 °C. However, the enzyme is denatured with urea and guanidine hydrochloride. The loss of activity is proportional to the urea concentration, the nondenatured enzyme being responsible for the remaining activity. Inactivation by urea is partially reversible. With urea or 60 °C heat denaturation, the enzyme dissociates into two types of subunits as revealed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Thus, α-galactosidase is the first external enzyme from yeast in which an oligomeric structure is reported. The enzyme catalyzes the hydrolysis of p-nitrophenyl-α-d-galactoside, melibiose, and raffinose with similar pH optima and V_max. However, the affinity is 20-fold lower for raffinose than for the other two substrates. Sugars having the same configuration in carbons 2, 3, and 4 as galactose competitively inhibit the enzyme.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

A rapid and sensitive paper electrophoresis assay for the detection of microbial siderophores elicited in solid-plating culture

A rapid and sensitive assay for the detection of microbial siderophores (iron-binding compounds) is described. Nine representative fungal and bacterial cultures including Ustilago sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae, Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by sequential transfers on iron-deficient solid-plating media. In response to Fe-stress conditions, the microorganisms excreted siderophore compounds into the extracellular solid culture medium. The solid agar matrix effectively concentrated and restricted the migration of the siderophore compounds to the region immediately adjacent to colonial growth. Agar-block samples from this region were removed and placed at the origin of an electrophoresis paper strip. The resultant absorbed material from the agar-block sample was subjected to high-voltage paper electrophoresis which separated the siderophore compounds by size and molecular net charge. Phenolic acid (“catechol”)-type siderophores were detected by fluorescence under uv light. Hydroxamic acid-type siderophores were visualized by spraying the electrophoretogram with ferric iron solution.     

The hemolytic plaque assay: theory for finite layers.

We extend the mathematical theory of hemolytic plaque growth to include plaques produced by cells secreting antibodies in layers of finite thickness. Previous theories have assumed that the layer was either two-dimensional or of infinite thickness. By using the method of images we derive an equation for the plaque radius as a function of time for layers of any thickness. We show that at short times and at long times the equation reduces to the appropriate infinite three-dimensional and two-dimensional limiting forms, and obtain expressions for estimating the range of times for which these limiting results are valid. For the liquid monolayer technique we obtain a new limiting result. The equation for the plaque radius is a transcendental equation which we solve numerically for a number of cases of interest. These results illustrate a variety of different features of plaque growth associated with the finite thickness of the layer. Experimental studies are usually carried out in layers whose thicknesses are not standardized. In the assays commonly used the thickness h can vary more than six hundred fold, i.e. 1 × 10^?3 cm ?h? 6.5 × 10^?1 cm. Such variation in h will cause widely different kinetics of plaque growth. For typical plaque experiments of one hour duration the two-dimensional limit is valid when h ? 3 × 10^?3 cm while the infinite thickness limit is valid when h? 10^?1 cm. For thicknesses in between these values the finite layer results must be used.     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

A new assay for L-aspartate:2-oxoglutarate aminotransferase.

A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l-glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l-glutamate and oxalacetate and the amino acceptor specificity using l-glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.     

Methylated cycloamyloses (cyclodextrins) and their inclusion properties

2,3,6-Tri-O-methyl and 2,6-di-O-methyl derivatives of cyclohexa- and cyclo-hepta-amylose (2,3,6-tri-O-methyl- and 2,6-di-O-methyl-α- and -β-cyclodextrin) have been prepared and shown to be versatile complexing agents. Their complexes in aqueous solution are usually more stable than the corresponding complexes of un-substituted cycloamyloses. The methylated cycloamyloses also form crystalline complexes, the stability of which depends on the size and shape of the guest molecule. The decomposition temperature of the crystalline complexes with homologous n-alkanes, which is relatively high increases with increasing chain-length of the hydro-carbon. The shift of the i.r. carbonyl band of oleic acid in its solid complex with methylated α-cyclodextrin probably reflects inclusion of the fatty acid in the mono-meric form. The methylated cycloamyloses, when used as the stationary phases for g.l.c. or dissolved in a conventional stationary phase, affect the retention times of organic compounds in a manner which suggests that inclusion phenomena are operative.     

Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase

The hepatotoxin thiobenzamide is S-oxidized by the microsomal flavin-containing monooxygenase (MFMO)¹ in liver, lung, and kidney of rabbit, mouse and rat. Its oxidation is accompanied by a large spectral shift which can be used as the basis of a simple convenient photometric assay for the MFMO system.     

Dissociation and oxygen equilibrium properties of earthworm (Pheretima hilgendorfi) hemoglobin

The dissociation and oxygen equilibrium properties of the purified hemoglobin and whole blood obtained from the earthworm Pheretima hilgendorfi were compared. Above pH 8.0, P1/2's were higher in the purified hemoglobin than in whole blood, while below pH 8.0, nearly identical P1/2's were observed in both materials and P1/2 was at a maximum at pH 6.5. The values of n1/2 were higher in whole blood than in the purified hemoglobin at alkaline pH. The maximum values of n1/2 were observed around pH 8.1 in the purified hemoglobin and pH 8.7 in whole blood, and the values were 5.3 and 9.5, respectively. In the purified hemoglobin, a small amount of dissociation component was already observed at pH 8.0, while in whole blood, no dissociation occurred up to pH 9.1. Dialysis of whole blood or addition of 10 mM EDTA to whole blood at alkaline pH induced the loss of the enhanced cooperativity, the increased oxygen affinity and the high stability of the 60 S whole molecule. These results strongly suggest that divalent cations are participating in the functional and dissociation properties of the whole blood of this species.     

The isolation and properties of porcine ferritin and apoferritin.

Porcine ferritin and apoferritin were purified to a greater degree of homogeneity than has been reported previously. Porcine ferritin was insoluble in the absence of a reducing agent, possessed a high content of iron, with an average $\text{Fe}\text{N}$ ratio of ~2.5, and contained almost no detectable endogenous apoferritin. The amino acid composition, ultraviolet-absorption spectrum, and ultraviolet-circular dichroism spectrum of porcine apoferritin are very similar to the respective parameters of equine apoferritin. The native and subunit molecular weights of porcine apoferritin are 503,000 and 20,000, respectively.     

The reduction of hydroxamic acids with titanium(III) chloride: a tool for the characterization of siderophores

Hydroxamic acid siderophores were observed to be inactivated by exposure to titanium(III) chloride. To study the reaction, a series of eight model hydroxamic acids were prepared and reacted with titanium(III) chloride. The products were shown by ir and NMR comparisons with authentic compounds to be the corresponding amides. The reduction was found to require 2 mol of titanium(III) per mol of hydroxamic acid.     

Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae.

Growth pH markedly influenced the composition of the cell envelope of $\text{Neisseria gonorrhoeae}$. The composition of the peptidoglycan from cells grown at pH 7.2 and 8.0 consisted primarily (91%) of muramic acid, glutamic acid, alanine, $\text{meso}$-diaminopimelic acid, and glucosamine in approximate molar ratios of 1:1:2:1:1. The peptidoglycan from cells grown at pH 6.0 contained an accessory protein(s) which accounted for 42% of the weight of the isolated complex.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.