Comparative studies on blood serum alpha-L-fucosidases from several mammalian species.

1. Peripheral blood serum alpha-L-fucosidases have been studied from various mammalian species: Sus scropha var domestica L. (pig), Capra hircus L. (goat), Bos taurus L. (bull, races Morucha and Charolais), Equus caballus L. (horse) and Equus asinus L. (donkey). 2. Fluorimetric and spectrophotometric procedures were used for determination of alpha-L-fucosidases. 3. alpha-L-Fucosidases were more active towards fluorescent substrates than towards chromogenic substrates. 4. pH optima values of the enzymes are: (A) 5.5 for sera from all above-mentioned species when fluorescent substrates were employed; (B) 4.0 for goat, 4.5 for bull, 5.0 for pig and 4.5-5.0 for horse and donkey sera when chromogenic substrates were used. 5. pH activity profiles are very similar for two races (Morucha and Charolais) of the same species (Bos taurus L.) and also for two species of the same genus (Equus caballus and Equus asinus L.). 6. These serum alpha-L-fucosidases are very labile under heat treatment, even at 37 degrees C.     

Comparative studies on six blood serum glycosidases from several mammalian species.

1. Peripheral blood serum alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, beta-D-xylosidase and beta-D-glucuronidase have been studied with a comparative point of view from several mammalian species: Bos taurus L. (bull), Capra hircus L. (goat), Sus scropha var. domestica L. (pig) and man. 2. Fluorimetric and spectrophotometric procedures were used for determination of enzyme activities and pH optima. 3. Glycosidase activity was generally higher with fluorescent substrates than with chromogenic substrates. 4. alpha-D-mannosidase was the most active with both fluorescent and chromogenic substrates. 5. All the studied enzymes had the same pH optimum (4.0) when the chromogenic substrates were used. 6. pH optima of these glycosidases ranged from 3.0 to 5.5 when the fluorescent substrates were used.     

Absence of tyrosine aminotransferase multiple forms in several mammalian animals

Tyrosine aminotransferase multiple forms occurring in rat liver are not present in all mammalian species. Among animals examined only rat and mouse liver possesses multiple forms of tyrosine aminotransferase; in guinea-pig, rabbit, bovine and sheep liver the enzyme occurs in a single form. The presence of lysosomal converting factor (cathepsin T), responsible for arising of multiple forms of tyrosine aminotransferase in rat liver, has been checked in another species lacking enzyme subforms. Lysosomal extracts of guinea-pig liver interconverts tyrosine aminotransferase from rat liver; lysosomal extracts of rat liver does not generate multiple forms of the enzyme from guinea-pig liver. It has been concluded that in some animals hepatic tyrosine aminotransferase is resistant to the proteolytic cleavage by lysosomal cathepsin T.     

The interaction of cefotaxime with the serum albumin of several mammalian species.

1. The interaction of cefotaxime with the serum albumin of several mammalian species; horses, swine, sheep, dogs and rabbits, was studied comparatively. The technique of ultrafiltration and spectrophotometric determination of the free antibiotic in the filtrate was used. 2. Binding percentages, which vary according to the species studied, were found to be higher in swine and rabbit albumins (between 92 and 81%) and lower for sheep, dog and horse albumins (between 67 and 52%). 3. The number of binding sites is usually close to 2; in the case of the horse it is 2.43. The apparent binding constants are: swine, 1.61 x 10(4) M-1; rabbit, 1.19 x 10(4) M-1; sheep, 2.33 x 10(3) M-1; dog, 2.00 x 10(3) M-1; horse, 1.42 x 10(3) M-1. The Scatchard model was used for data analysis. 4. Possible consequences of this interaction regarding clinical use of cefotaxime on different species are discussed.     

A study of the adhesive glycoprotein-inactivating protein from mammalian blood serum

A protein with a molecular weight of 70 kDa was isolated from bovine blood serum and purified to a homogenous state. This protein inhibited reversibly the adhesive serum glycoprotein with a molecular weight of 12 kDa, which displayed biological activity at ultralow doses. Amino acid analysis showed that the protein inactivator belongs to the group of prealbumins from vertebrate blood serum. The secondary structure of its molecule was characterized by a considerable number of alpha-helices. The conditions for inactivation of serum glycoprotein were studied. The interaction between the serum glycoprotein and the protein inactivator occurred over a long period of time (1 day). It should be emphasized that the presence of calcium ions was a necessary condition for the inactivation of the serum glycoprotein. The data suggest that inactivation of serum glycoprotein results from the formation of a molecular complex consisting of the protein inactivator and the glycoprotein, which is related to the carbon-protein interaction.     

Effect of chloride and diamide on serum angiotensin i-converting enzyme activity from eight mammalian species

1. The effect of chloride on serum angiotensin I-converting enzyme (ACE) activity was characterized in eight mammalian species: dog, guinea pig, hamster, human, mouse, rabbit, rat, and sheep.2. Optimum chloride concentrations varied from 300 mM for rabbit to 1700 mM for hamster.3. The increments with these optimum concentrations with respect to 100 mM chloride concentration were from 1.4-fold in rabbit to 7.9-fold in hamster and dog.4. There was no correlation between serum chloride concentration or serum ACE activity and optimum chloride concentration.5. Serum ACE increased only in humans with diamide pretreatment suggesting the presence of endogenous inhibitors.     

Evidence for different glycohydrolase and glycosyltransferase activities of beta-N-acetylglucosaminidases A and B.

1) Two forms of beta-N-acetylglucosaminidase--known as form A and form B - were purified from bovine spleen homogenates and efficaciously separated by preparative disc electrophoresis on polyacrylamide gel. Studies on the enzymatic specificity revealed that the two forms have different glycoside hydrolase and glycosyl transferase activities towards substrates of natural origin. 2) With the trisaccharide GlcNAc-GlcUA-GlcNAc from hyaluronate as substrate, form A released free N-acetylglucosamine at a rate 35-40 times higher than form B. The B form, however, transferred N-acetyl-[6-3H]glucosamine from phenyl-beta-N-acetyl-D[6-3H]glucosaminide to the tetrasaccharides GlcUA-GalNAc-4-sulfate-GlcUA--GalNAc-4-sulfate or GlcUA-GlcNAc-GlcUA-GlcNAc isolated from chondroitin 4-sulfate or hyaluronate at rates 5-10 times higher than beta-N-acetyl-glucosaminidase A, the corresponding 3H-pentasaccharides being isolated as reaction products. 3) The pH optimum of the glycoside hydrolase activity is 4.5, while optimum glycosyl transfer proceeds at pH 6.5. Under condition optimum for glycoside transferase, hydrolytic activity is still observed with each form, but the B form exhibits about equal glycoside hydrolase and glycoside transferase activity, whereas the A form has a predominant glycoside hydrolase action.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.     

Quaternary structure of oligomeric immunoglobulin A forms from human blood serum]

A character of forces stabilyzing quaternary structure of dimer and more high molecular human immunoglobulin A oligomers is found to be different. Quaternary structure of IgA dimer is formed when joining subunits with disulfide bonds and is stabilized by non-covalent interactions between them. Disulfide bonds play a main part in the formation of trimers and tetramers. Dimer IgA reconstructs by 40% from subunits with intact interchain S--S bonds. The addition of exogenous J-chain does not significantly affect the process of dimer self-assembling from subunits with recovered and intact interchain disulfide bonds.     

Fluorescence polarization studies of different forms of angiotensin-converting enzyme

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied by the fluorescence polarization technique. The dissociation constants K _d of the enzyme-inhibitor complexes in 50 mM Hepes-buffer, pH 7.5, containing 150 mM NaCl and 1 M ZnCl₂ at 37°C were (2.3 ± 0.4)·10^–8, (2.1 ± 0.3)·10^–8, and (2.1 ± 0.2)·10^–8 M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 ± 0.2)·10^–3 M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 ± 0.01) sec^–1 and (0.026 ± 0.001) sec^–1 that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 ± 0.001) and (0.041 ± 0.001) sec^–1 for the N-domain and for testicular ACE, respectively.     

Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.