[Changes in the kinetic characteristics of reactions catalyzed by erythrocyte delta-aminolevulinate dehydratase in experimental lead poisoning in rats]. / Izmenenie kineticheskikh kharakteristik reaktsii, kataliziruemoi delta-aminolevulinatdegidratazoi éritrotsitov, pri éksperimental'no svintsovoi intoksikatsii u krys.

An increase in values of both kinetic parameters (Km and Vmax) of the delta-aminolevulinate dehydratase reaction was observed in blood of rats poisoned with lead. The Km value of the reaction was increased, while Vmax--decreased, after addition of Pb2+ into rat blood in vitro. The results obtained as well as the data published in literature suggest that a compensatory mechanism appears to function in vivo, where a substrate, delta-aminolevulenic acid, plays a triggering role.

[Various regulatory properties of fructose-1,6-diphosphatase of the rat liver under normal conditions and in experimental autoimmune cardiomyopathy]. / Nekotorye reguliatornye svoistva fruktozo-1,6-bifosfatazy pecheni krysy v norme i pri éksperimental'noi autoimmunnoi kardiomiopatii.

An increase in fructose-1,6-biphosphatase activity in liver tissue of rats with experimental autoimmune cardiomyopathy was observed. At certain concentrations of EDTA and Mg2+ the AMP-inhibition curves exhibited an intermediate plateau, which appear under different experimental, normal and pathological conditions. The occurrence of complex kinetic curves could be attributed to simultaneous existence of several enzymatic forms differing in the "structural" sites with tightly bound metals, which defines the differences in the kinetic cooperativity and sensitivity to AMP inhibition.

[Desensitization with acetylsalicylate and salicylate of fructose-1,6-diphosphatase from the rabbit liver and skeletal muscles to allosteric inhibition of AMP]. / Desensibilizatsiia atsetilsalitsilatom i salitsilatom fruktozo-1,6-bifosfatazy iz pecheni i skeletnykh myshts krolika k allostericheskomu ingibirovaniiu AMF.

Acetylsalicylate and salicylate partially desensitized fructose 1,6-biphosphatase from rabbit liver and skeletal muscle to allosteric AMP inhibition. The desensitization was accompanied by a decrease in cooperativity between allosteric sites especially distinct for the liver isoenzyme. The effect of salicylate on both isoenzymes was more pronounced than that of acetylsalicylate. No essential differences in the effect of the compounds studied on these isoenzymes were found.

[Complex type of kinetics of fructose-1,6-diphosphate from the rat and rabbit liver]. / Slozhnyi kharakter kinetiki fruktozo-1,6-bifosfata iz pecheni krysy i krolika.

Studies on rat and rabbit liver fructose 1.6-bisphosphatase inhibition by AMP showed that with an increase in EDTA concentration the hyperbolic AMP inhibition curve is transformed into a sigmoidal one. At intermediate EDTA concentrations, the kinetic curves have a plateau. The appearance of the intermediate plateau may be due to the superposition of kinetic curves corresponding to two enzyme forms simultaneously present in the assay mixture. One of these forms deprived of endogenous Me2+ (presumably Zn2+) is inhibited by AMP in a cooperative manner, while the other one retains Me2+ which prevents the cooperative response of the enzyme to AMP.

[Kinetic and allosteric properties of highly-purified, biosynthetic L-threonine dehydrogenase from brewer's yeast Saccharomyces carlsbergensis]. / Kineticheskie i allostericheskie svoistva vysokoochishchennoi "biosinteticheskoi" L-treonindegidratazy iz pivnykh drozhzhei Saccharomyces carlsbergensis.

Kinetic and allosteric propeties of highly purified "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlbergensis were studied at three pH values, using L-threonine and L-serine as substrates. It was shown that the plot of the initial reaction rate (v) versus initial substrate concentrations ([S]0 pH 6.5 is hyperbolic (Km=5.0.10-2M), while these at pH 7.8 and 9.5 have a faintly pronounced sigmoidal shape with fast occurring saturation plateaus ([S]0.5= 1.0.10-2 and 0.9.10-2M, respectively). the ratios between L-threonine and L-serine dehydratation rates depend on pH. The kinetic properties and the dependence of substrate specificity on pH suggest that the enzyme molecule undergoes pH-induced (at pH 7.0) conformational changes. The determination of pK values of the enzyme functional groups involved in L-threonine binding demonstrated that these groups have pK is approximately equal to 7.5 and 9.5. The latter group was hypothetically identified as a epsilon-NH2-group of the lysine residue. High concentrations of the allosteric inhibitor (L-isoleucine) decrease the rates of L-threonine and L-serine dehydratation and induce the appearance (at pH 6.5) or increase (at pH 7.9 and 9.5) of homotropic cooperative interactions between the active sites in the course of L-threonine dehydratation. The enzyme inhibition by L-isoleucine increases with a decrease of L-threonine concentrations. Low L-isoleucine concentrations, as well as the enzyme activator (L-valine) stimulate the enzyme at non-saturating substrate concentrations (when L-threonine or L-serine are used as substrates) without normalization of (v) versus [S]0 plots. The maximal activation of the enzyme is observed at pHG 8.5--9.0. It is assumed that the molecule of "biosynthetic" L-threonine dehydratase from brewer's yeast contains two types of sites responsible for the effector binding, i.e., "activatory" and "inhibitory" ones.

Purification of commercial preparations of NADP+ from AMP contamination.

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.

[Purification of commercial preparations of NADP from AMP]. / Ochistka kommercheskikh preparatov NADF ot AMF.

A method in described for elimination of AMP from commercially available preparations of NADP. The method enables to obtain desalted NADP preparations of 98-99.5% purity with a yield of 70-80% using only one chromatographic step. The procedure included anion exchange chromatography on Dowex 1, HCOO- -form. This chromatographic step completely separated AMP from NADP in a gradient of HCOOH concentrations from 0 to 2 N, and NADP was eluted by 1.5 N HCOOH containing 0.15 N HCOOK; during subsequent precipitation and washing of NADP by means of acetone HCOOK remained in a solution. Addition of HCOOK was required for a decrease in NADP elution volume which was important for the subsequent precipitation with acetone.

[Regulatory enzymopathies]. / Reguliatornye énzimopatii.

Hereditary regulatory enzymopathies are considered, molecular-genetic mechanism of which involved a mutation of a structural gene in the locus, coding the much less than regular much greater than amino acid sequence in the allosteric site of regulatory enzyme or the site of the enzyme polypeptide chain, which is responsible for allosteric conformational transition. Dissimilar manifestations of regulatory and classical enzymopathies are discussed. Estimation of kinetic patterns might be very useful in diagnosis of regulatory enzymopathies as shown during study of some molecular pathologies, which occurred, for example, due to molecular pathology of phosphofructokinase and phosphoribosyl pyrophosphate synthetase. Impairment of allosteric regulation of L-threonine-L-serine dehydratase is considered as a model of regulatory enzymopathy, found in spontaneous hepatomas of highly carcinogenous CBA mice strain. Treatment of regulatory enzymopathies is considered depending on the effect of well known chemotherapeutic drugs (mainly, acetyl salicylic acid) on allosteric functions of regulatory enzymes acetyl-CoA-carboxylase and fructose-1,6-bisphosphatase from animal liver tissue.

[Certain properties of "biosynthetic" L-threonine dehydratase from subcellular structures of brewers' yeast Saccharomyces carlsbergensis]. / Nekotorye svoistva "biosinteticheskoi" L-treonindegidratazy iz subkletochnykh struktur drozhzhei Saccharomyces carlsbergensis.

The paper is concerned with kinetic properties of the "biosynthetic" L-threonine dehydratase (EC 4.2.1.16) solubilized from subcellular structures of brewers' yeast Saccharomyces carlsbergensis in the absence and presence of the allosteric inhibitor, L-isoleucine, at three pH-values (pH 6.5, 7.8 and 9.5). The curve of the initial reaction rate versus initial substrate concentration in the absence of L-isoleucine at pH 6.5 was of hyperbolic character (Km = 5.5.10(-2) M), and at pH 7.8 and 9.5 the kinetic curve had a weakly sigmoidal pattern with a sharp going into the saturation plateaux; the values of [S] 0.5 are 1.10(-2) and 8.7.10(-3) M, respectively. An addition of L-isoleucine to the reaction mixture led to the appearance (at pH 6.5) or to an increase (at pH 7.8 and 9.5) of the sigmoidality of these kinetic curves and to a decrease in values of the maximum reaction rate V. The enzyme sensibility to the inhibitory effect of L-isoleucine decreased with an increase in pH values. Low L-isoleucine concentrations at low substrate concentrations activated the enzyme. The pH optimum for L-threonine dehydratase under study was 9.5-10.0. The enzyme molecular weight is about 300 000.

[Comparative properties of multiple forms of rabbit muscle phosphofructokinase]. / Sravnitel'naia kharakteristika mnozhestvennykh form fosfofruktokinazy iz myshts krolika.

Some properties of three interconvertible forms of rabbit muscle phosphofructokinase specifically eluted from DEAE-cellulose with 19 mM citrate in 0.1 M tris-phosphate buffer, pH 8,0 (I), with 0,3 M buffer (II) and 1.5 M NaCl (III) are compared. Forms I-III differ in specific activities, alpha-helices content and sedimentation properties. The kinetic behaviour of forms I and III in 25 mM glycylglycine-beta-glycerophosphate, pH 8.3, at inhibitory ATP concentrations is characterized by biphasic velocity versus fructose-6-phosphate concentration curves with nH = 1.0 and 2.3, but with different V and [S]0.5 for the respective forms. At pH 6.8 from I is characterized by the kinetic curves with a lag period, while form III--by that with a burst. Form I reveals negative cooperativity in initial and stationary velocities at low substrate concentrations. The stationary velocity of form III is characterized by negative cooperativity within the whole concentration range studied. At pH 7.0 both forms are inhibited by citrate according to the initial and stationary velocities; however, the Ki values are different. The complex kinetic behaviour of phosphofructokinase corresponds to its complex chromatographic and sedimentation behaviour. The multiplicity of the enzyme forms seems to be due to a complex set of its oligomers and conformers and a hysteretic type of transitions between them as well as to its phosphorylation and possible binding of ligands.

[Choice of the association model of rabbit muscle phosphofructokinase using mathematical modeling]. / Vybor modeli assostiatsii fosfofruktokinazy iz myshts krolika metodom matematicheskogo modelirovaniia.

The sedimentation behaviour of the subform of rabbit muscle phosphofructokinase specifically eluted from DEAE cellulose by citrate was studied in different media by velocity experiments. The measured sedimentation coefficients of different components in the system can be classified into 12 groups, which is indicative of a complex multistep association process of the enzyme (with more than 3 oligomers). The concentration dependence of weight average sedimentation coefficient of phosphofructokinase was a studied. The choice of the probable association model of phosphofructokinase oligomers at low protein concentration was accomplished by means of computer simulation of the association process. Of 26 closed association models tested 14 models with 2 or 3 association constants are indistinguishable in view of Student's t-criterion of significance. All uniparametric association models studied significantly inferior approximate the experimental data. It is supposed that the dimer is not the structural unit of polymerization. Having this in mind and taking into account the demand for the multistep process, one may consider as most probable only 8 models of phosphofructokinase association, namely monomer-dimer-trimer-tetramer-monomer-dimer-tetramer-octamer (with 3 association constants) and linear polymerization up to the octamer with 2 association constants, where the "monomer" of association may be defined as trimer, tetramer or hexamer of subunits.

[Comparative kinetic properties of structure-bound and solubilized L-threonine dehydratase from brewer's yeast]. / Sravnenie kineticheskikh svoistv strukturnosviazannoi i soliubilizirovannoi L-treonindegidratazy iz pivnykh drozhzhei.

It has been shown that L-threonine dehydratase (EC 4.2.1.16) of brewer's yeast Saccharomyces carlsbergensis is localized in the mitochondrial fraction. The enzyme is easily solubilized from the mitochondria by changing the pH and ionic strength of the buffer. Some kinetic properties of structure-bound and solubilized L-threonine dehydratase have been compared at pH 6,5. The kinetic plots of the initial rate of the reaction versus initial substrate concentration for both enzymes have a hyperbolic shape; the affinities of both enzymes for the substrate appear to be similar (Km = 20 mM). Both enzymes are inhibited by L-isoleucine, the shape of the kinetic plots being thereby changed into sigmoidal. Solubilization results in a decrease of the mitochondral enzyme sensitivity to the inhibition by L-isoleucine and in an appearance of cooperative interactions between the allosteric sites.

[Kinetic patterns of their reactions catalyzed by membrane-bound monoamine oxidase]. / Osobennosti kinetiki reaktsii kataliziruemykh membranosviazannymi monoaminoksidazami.

The initial rate (v) versus initial substrate concentration [S]0 plots for the reaction of serotonin deamination catalyzed by rat liver mitochondrial monoamine oxidase are characterized by complicated non-hyperbolic patterns. The shapes of the curves in the plots depend on the age and strain of the animals used or on alterations in the physico-chemical state of mitochondria caused by freezing -- thawing and swelling in hypotonic solutions. The v versus [S]0 plots for other substrates (e. g. tyramine) do not obey the Michaelis -- Menten equation as well. In order to interpret the kinetic patterns described a model of the mitochondrial monoamine oxidase structure and function has been proposed. The model is based on the assumption that both catalytic and regulatory allosteric sites are present on the surface of monoamine oxidase molecules. In the process of serotonin or tyramine binding the monoamine oxidases exhibit cooperative properties which are characteristic for the allosteric enzymes. The model also suggests an active regulatory role of the mitochondrial membrane itself.

[Formation of higher alcohols by Saccharomyces carlsbergensis from branched-chain amino acids and their keto analogs]. / Obrazovanie vysshikh spirtov Saccharomyces carlsbergensis iz razvetvlennykh aminokislot i ikh ketoanalogov.

A new method of chemical synthesis of alpha-ketoisocaproic acid (the keto analogue of L-leucine) is described. It has been shown that the resting cells of Saccharomyces carlsbergensis 776, in the stationary state of biomass, produce mainly higher alcohols: isobutanol from L-valine and its keto analogue; optically active amylol only from L-isoleucine and its keto analogue; isoamylol only from L-leucine and its keto analogue. "Nonspecific" formation of n-propanol from L-valine, L-isoleucine and their keto analogues, as well as that of isobutanol from L-isoleucine and its keto analogue, has been also found at pH 7.0. Formation of higher alcohols from alpha-keto acids has an acidic pH optimum while that from L-amino acids has a neutral or a weakly alkaline pH optimum. Formation of isobutanol from L-valine is an exception. The dependence of higher alcohol formation on the pH and the kinetics of their accumulation suggest that higher alcohols are produced from L-amino acids in at least three sequential reactions: transamination, decarboxylation of the keto analogue being formed, and reduction of the aldehyde; formation of higher alcohols from alpha-keto acids involves two reactions: decarboxylation and reduction. Transamination and decarboxylation are limiting steps in the process in the former case, and decarboxylation in the latter.

[Effect of higher alcohols on the activity of biosynthetic L-threonine dehydratase from Saccharomyces carlsbergensis brewer's yeast]. / Vliianie vysshikh spirtov na aktivnost' biosinteticheskoi L-treonindegidratazy iz pivnykh drozhzhei Saccharomyces carlsbergensis.

The effects of low (1 . 10(-4) M) and high (1 . 10(-3) M) concentrations of n-propanol, isobutanol and isoamylols on the kinetic behaviour of "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlsbergensis 776 were studied. It was concluded that these alcohols control the activity of the first enzyme of the L-threonine biosynthetic pathway.

[Kinetics and thermodynamics of heat inactivation of L-threonine-L-serine dehydratase from human liver]. / Kinetika i termodinamika teplovoi inaktivatsii 8treonin-L-serindegidratazy iz pecheni cheloveka.

It has been shown that heat inactivation of L-threonine- and L-serine dehydratase activities at 37; 45; 50 and 55 degrees C in human liver extracts (the liver was ectracted with buffer containing 1.10(-5) M of pyridoxal 5'-phosphate) in course of time is practically identical, and characterizes by the same of values of activation energy of heat inactivation process, activation enthalpy of this process, activation free energy of that and activation enthalpy of the heat inactivation process. A rise of pyridoxal 5'-phosphate concentration (to 2.10(-4) M) in the buffer used for the liver extraction and hence in the medium in which the heat inactivation process was carried out stabilises L-threonine- and L-serine dehydratase activities against the inactivation at 55 degrees C. It has been concluded thatL-threonine- and L-serine dehydratase activities in human liver belong to the single protein or to two proteins having very like physico-chemical properties, and that pyridoxal 5'-phosphate is essential for this enzyme not only as coenzyme but also it is necessary to support active and stable conformation of this oligomeric protein.

[Detection and properties of L-threonine-L-serine dehydratase in human liver]. / Obnaruzhenie i svoistva L-treonin-L-serindegidratazy pecheni cheloveka.

It has been shown that in liver extract of men deceased by different causes, L-threonine and L-serine dehydratase activities probably, belonging to only one enzyme--L-threonine-L-serine dehydratase--are found. Both activities and their ratios depend on K+ concentration both in the buffer used for enzyme extraction and in the reaction medium. Before extraction of active and stable forms of enzyme the liver is to homogenized in a buffer containing 0.15 M KCl. Both enzymatic activities have a pH-optimum at pH 9.6--10.0. It was shown that D-isomers of threonine and serine are not dehydratated and do not inhibit dehydratation of L-isomers. Studies of dependence of L-threonine and L-serine dehydratase reaction rates on temperature showed that at any temperature ranges the energy activation values are higher for the L-threonine dehydratase reaction than for the L-serine dehydratase reaction and that the ratio reaction rates for both reactions depends on temperature.

[Characteristics of L-threonine- and L-serine dehydratases from mouse liver and spontaneous hepatomas]. / Nekotorye osobennosti L-treonin- i L-serindegidratazy pecheni myshei i spontannykh gepatom.

High activities of L-threonine and l-serine dehydratases were maintained in spontaneous hepatoma of mice strain CBA as distinct from transplantable hepatoma. The initial rate [v] versus substrate concentration [S]0 curves had complex shapes for the enzymes from the liver tissue of healthy animals (especially after extraction of the enzymes by means of buffers with low concentration of K+). Kinetic patterns of l-threonine and L-serine dehydratases from hepatoma were distinct from those of normal mice liver tissue. The shape of [v] versus [S]0 plots was altered in response to AMP (1.10(-3) M). Contrary to the enzymes from normal tissue, dehydratases from hepatoma did not alter the shape of [v] versus [S]0 curves in response to AMP. The enzymes from hepatoma were apparently desensitized with respect to their possible allosteric effector. Threonine dehydratases of normal mice liver were also dissimilar as compared with the enzymes from hepatoma in the affinity of the apoenzyme to pyridoxal 5'-phosphate. This affinity was 3-fold lower for threonine dehydratase from hepatoma as compared with the enzyme from liver tissue.