[Characteristics of rat ceruloplasmin from the serum of animals, which received salts of silver with food].

Abiogenic Ag(I) ions have electronic structure, similar to Cu(I) ions and can compete with Cu(I) for binding sites of proteins which transport copper from extracellular media to sites of cuproenzyme formation in the cell. Rodents receiving Ag-salts with food develop extracellular deficiency of copper associated with ceruloplasmin (Cp, the major copper-transporting protein in blood serum of vertebrates). The present work focuses on the studies of biochemical and physicochemical properties of Cp, obtained from blood serum of rats, which received AgCl with food for 4 weeks (Ag-rats). Cp-fractions from blood serum of Ag-rats (Ag-Cp) were obtained by ion-exchange chromatography with stepped gradient of NaCl. Each fraction was tested for oxidase and ferroxidase activities by direct measurement of catalytic activity in the gel, and for specific activity in holo-Cp in oxidation of chromogenic substrate. Molecular mass, electrophoretic mobility and ratio of apo- and holo-forms in Ag-Cp fractions were evaluated by immunoblotting. Ag-Cp samples did not contain products of spontaneous partial proteolytic degradation, characteristic of holo-Cp samples. Fractions of Ag-Cp and holo-Cp (from blood serum of control rats) were compared by optical spectra, tertiary structure, susceptibility to thermal denaturation, and by atomic Cu and Ag content. Ag-Cp contained 1-2% Cp, which is similar by spectral and catalytic properties with holo-Cp. [Ag]:[Cu] ration in Ag-Cp samples was about 4:1. As evidenced by circular dichroism and differential scanning calorimetric studies, the major apo-fraction of Ag-Cp lacked tertiary structure of native Cp and was significantly misfolded, which might explain its resistance to spontaneous partial proteolytic degradation.

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk.

BACKGROUND: New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS: The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS: All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS: IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

Transglycosylation activity of alpha-D-galactosidase from Trichoderma reesei. An investigation of the active site.

The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.

[Fungal aspartic proteinase from Trichoderma viride. Specificity during oligopeptide hydrolysis]. / Gribnaia aspartatnaia proteinaza iz Trichoderma viride. Spetsifichnost' pri gidrolize oligopeptidov.

We isolated, purified, and characterized an aspartic protease from fungus Trichoderma viride. The pH-dependence of the enzyme functioning was determined, and its specificity in the limited proteolysis of insulin and melittin was compared to the specificities of pepsin A and gastricsin. The kinetics of melittin hydrolysis by these enzymes was studied by mass spectrometry.

[Effect of modifications of a series of amino acid radicals on the enzymatic activity of glucoamylase from Aspergillus awamori]. / Vliianie modifikatsii riada aminokislotnykh radikalov na fermentativnuiu aktivnost' gliukoamilazy iz Aspergillus awamori.

The effect of chemical modification of various amino acid residues on the enzymatic activity of glucoamylase from Asp. awamori was studied. Modification of the carboxyl groups by taurine in the presence of water-soluble carbodiimide results in complete inactivation of the enzyme. The inactivation process includes two steps, namely non-specific modification and modification of the active center carboxyls. The rate constants of inactivation at both steps were measured in the presence and absence of the substrate, i. e. maltose. It was shown that the enzyme is inactivated by N-bromosuccinimide. Based on the data on the protection of the enzyme active center by the substrates (maltooligosaccharides of various lengths), it was concluded that the essential tryptophane residue(s) is localized in the fourth subsite. Ethoxycarbonylation, nitration and acetylation of glucoamylase do not change the catalytic activity of the enzyme. The protein was shown to contain no SH-groups.

[Carboxyl groups in the active center of glucoamylase from Aspergillus awamori]. / Karboksil'nye gruppy v aktivnom tsentre gluikoamilazy iz Aspergillus awamori.

The values for the ionization constants of the catalytic groups of the active site of glucoamylase from Asp. awamori for the free enzyme and for the enzyme--substrate complex were calculated. The temperature dependence of the alkaline branch of the pH-dependence curve and the pH dependence in the presence of methanol were determined. The ionization enthalpy delta H = 1.5 +/- 0.3 kcal/mole, the ionization entropy delta S = 20.5 +/- 1.2 e. u. It was assumed that two carboxyl groups are involved in the catalytic act.

Crystallization and preliminary X-ray study of beta--mannosidase from Trichoderma reesei.

beta-Mannosidase from Trichoderma reesei, a 105 kDa glycoprotein, has been crystallized. The crystals belong to the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions a = b = 165.86, c = 122.46 A, and diffract beyond 2.75 A resolution. X-ray diffraction data were collected from a frozen crystal on a synchrotron X-ray source.

The action of alpha-mannosidase from Oerskovia sp. on the mannose-rich O-linked sugar chains of glycoproteins.

Alpha-mannosidase was isolated from the culture liquid of Oerskovia sp. The purified enzyme had a molecular mass of 480 kDa and comprises four identical subunits. The enzyme cleaves bonds in side chains of yeast mannan (Km = 0.08 mM, k(cat) = 1.02 micromol x min(-1) x mg(-1)) and reveals a low activity towards p-nitrophenyl alpha-D-mannopyranoside. The alpha-mannosidase is a Ca2+-dependent enzyme and is inhibited by EDTA. The enzyme possess no endo-mannosidase activity releasing only mannose in the reaction with the inversion of anomeric configuration and could be classified as exo-alpha-mannanase. The enzyme revealed a high deglycosylating activity towards the short mannose-rich O-linked carbohydrate chains of glycoproteins.

The carbohydrate moiety of alpha-galactosidase from Trichoderma reesei.

Alpha-galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by beta-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: alpha1-6 D-mannopyranosyl-alpha-D-glycopyranoside and alpha1-6 D-glucopyranosyl-alpha-D-galactopyranoside and one trimer: alpha-D-glucopyranosyl-alpha1-2 D-mannopyranosyl-alpha1-6 D-galactopyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo-beta-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo-beta-N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of alpha-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of alpha-galactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

alpha-Mannosidase from Trichoderma reesei participates in the postsecretory deglycosylation of glycoproteins.

The 160 kDa alpha-mannosidase (E.C. 3.2.1.24) isolated from culture filtrate of Trichoderma reesei has wide aglycon specificity but cleaves the alpha1 --> 2 and alpha1 --> 3 mannosidic bonds with higher rate than alpha1 --> 6 bond and slowly hydrolyses yeast mannan and 1,6-alpha-mannan. The specific activity of the enzyme and rate constant in the reaction with p-nitrophenyl-alpha-D-mannopyranoside were 0.15 U/mg and 1.62 x 10(-4) microM/min/microg, respectively, at optimal pH 6.5. We have found that in vitro enzyme is able to cleave off 30% of total alpha-mannopyranosyl residues from N- and O-linked glycans of secreted glycoproteins. The activity of the alpha-mannosidase toward glycoproteins in vivo was studied comparing the structures of O- and N-linked glycans of glycoproteins isolated from the cultures growing with and without 1-deoxymannojirimycin, an inhibitor of alpha-mannosidases. Difference in structures of these glycans may be explained by postsecretory deglycosylation catalysed by the alpha-mannosidase.

Isolation, enzymatic properties, and mode of action of an exo-1,3-beta-glucanase from Trichoderma viride.

An exo-1,3-beta-glucanase has been isolated from cultural filtrate of T. viride AZ36. The N-terminal sequence of the purified enzyme (m = 61 +/- 1 kDa) showed no significant homology to other known glucanases. The 1,3-beta-glucanase displayed high activity against laminarins, curdlan, and 1,3-beta-oligoglucosides, but acted slowly on 1,3-1,4-beta-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-beta-glucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration. Whereas only glucose was released from the nonreducing terminus during hydrolysis of 1,3-beta-oligoglucosides, transient accumulation of gentiobiose was observed during hydrolysis of laminarins. The gentiobiose was subsequently degraded to glucose. The Michaelis constants Km and Vmax have been determined for the hydrolysis of 1,3-beta-oligoglucosides with degrees of polymerization ranging from 2 to 6. Based on these data, binding affinities for subsites were calculated. Substrate binding site contained at least five binding sites for sugar residues.