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L V Belousova

Publications and source records attributed to L V Belousova.

At least 19 recordsLinked to original sources
PubMed2006

Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase.

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60 degrees C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A(lim)) is proportional to protein concentration at both temperatures. The first order rate constant (k(I)) does not depend on protein concentration in the range 0.05-0.2 mg/ml at temperature 50.6 degrees C, but at temperature 60 degrees C it increases with the growth of protein concentration in the range 0.1-0.4 mg/ml. Kinetic curves, shown in coordinates {A/A(lim); t}, in experiments at 50.6 degrees C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60 degrees C, half-transformation time t(1/2) = ln2/k(I) decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60 degrees C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6 degrees C aggregation rate is limited by the stage of protein molecule denaturation, but at 60 degrees C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation.

Animals↗
PubMed2004

Kinetics of chemical modification of arginine residues in mitochondrial creatine kinase from bovine heart: evidence for negative cooperativity.

The kinetics of chemical modification of arginine residues in mitochondrial creatine kinase (mit-CK) from beef heart by 4-hydroxy-3-nitrophenylglyoxal (HNPG) have been studied with simultaneous registration of enzyme inactivation. Experiments showed that complete inactivation of mit-CK corresponded to modification of two arginine residues per mit-CK monomer. The data on the modification kinetics can be described by the sum of two exponential terms and suggest strong negative cooperativity in the binding of HNPG to arginine residues. The rate constants for the fast and slow phases of modification differ by a factor of about 50. The corresponding rate constants for inactivation differ by a factor of about 30. The rate constant for the slow stage of inactivation is twice as large as that for the rate constant for the slow stage of modification, i.e., the inactivation process is ahead of the modification process.

Animals↗
PubMed1993

A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength.

The quantitative aspects of mitochondrial creatine kinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (Ka = 0.39 mM at ionic strength I = 7.5 mM and 5 degrees C); after protonation its affinity increased many times (Kah = 39 nM). Determination of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave Kah = 15 nM. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückel's theory for activity coefficients assuming the charges of the interacting species to be +4 and -4. The dissociation constant for the mitCK-membrane complex at pH 7.4 and I = 0 was evaluated by different approaches as approx. 1 nM. Extramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since in the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.

Adsorption↗
PubMed1991

The structural features of beef heart mitochondrial creatine kinase.

Two forms of mitochondrial creatine kinase (Mi-CK) having Mr 320 kDa and 240 kDa as determined by gel-filtration on Sephacryl S-300 in 0.1 M Tris-HCl pH 7.4 were investigated. The sedimentation coefficient values for these two forms were found to be identical and equal to 12.3 S. When studied by electron microscopy the main type of images for the 320 kDa and 240 kDa Mi-CK appeared as annular particles, 12-14 nm in diameter, with a well-detected subunit structure and a central hollow, 3-4nm in diameter filled with the dye. The results of the averaging of the main type of individual Mi-CK images and particles of the two-dimensional crystal layer point to the overall geometry of the Mi-CK molecule structure as containing eight subunits arranged by a 4-fold symmetry around the central hollow. It may be that the eight identical subunits of crystalline Mi-CK are arranged with a P422 symmetry. However in both cases the averaged main images do not show a mirror symmetry. The multiplicity of the observed projections close to annular one provides additional evidence in favour of the great lability and structural mobility of the Mi-CK subunits. It allows to assume that two forms (320 kDa and 240 kDa) are not the different oligomers but they are two functionally distinct conformational states of octameric molecule of Mi-CK.

Animals↗
PubMed1989

[Octameric mitochondrial and dimeric cytoplasmic creatine kinase. The number of subunits, participating in catalysis].

It was found that in the octameric form of mitochondrial creatine kinase (Mr = 340 kD), only 52% of active centers bind Mg-ADP into a E-Mg-ADP-creatine complex with the dissociation constant, K(Cr)ADP, of 0.105 mM, which is close to the Km value for the enzyme (0.072 mM). In the dimeric form of cytoplasmic creatine kinase (Mr = 82 kD), 100% of active centers bind Mg--ADP; the K(Cr)ADP value (0.11 mM) is close to the Km value for the given enzyme preparation (0.083 mM). All active centers of rabbit muscle cytoplasmic creatine kinase were shown to form an analog of the transition state complex (ATSC) - E-Mg-ADP-NO3- -creatine. The constant for Mg-ADP dissociation from ATSC is identical for all centers of cytoplasmic creatine kinase and equals to 6.0 microM. The curves for ATSC saturation with Mg-ADP in the presence of iodacetamide for mitochondrial creatine kinase were constructed and computer analyzed. It was shown that in the octameric form of the enzyme only 54 +/- 13% of subunits can form ATSC. The constant for Mg-ADP dissociation from ATSC, KATSCADP is equal to 1.9 +/- 0.8 microM. It was concluded that 50% of subunits of the octameric form of mitochondrial creatine kinase are not involved in the catalytic act due to masking of their active centres and their inability to form transition state complexes. A model of regulation of cell supply with high energy compounds, e.g., ATP, creatine phosphate, via association-dissociation of mitochondrial creatine kinase oligomers is proposed.

Adenosine Diphosphate↗
PubMed1988

[Effect of oligomerization on the properties of essential SH-groups of mitochondrial creatine kinase].

The properties of creatine kinase isolated from bovine heart mitochondria in dimeric (Mr = 84 +/- 6 kD) and octameric (Mr = 340 +/- 17 kD) forms were compared with those of the earlier described hexameric form of the enzyme (Mr = 240 +/- 12 kD). The kinetics of SH-group modification by DTNB, the inactivation kinetics as well as the number of modified SH-groups point to significant differences between the three oligomeric forms of the enzyme. Each subunit of creatine kinase was found to possess one "fast" essential cysteine residue whose modification by DTNB and iodoacetamide led to enzyme inactivation. The formation of an analog of the transition state complex (E--MgADP--NO3--creatine) was paralleled with partial protection of only the "fast" cysteine residue which manifested itself in the decrease of the rate of its interaction with DTNB in all the three oligomeric forms. Dimer association into a hexamer and octamer occurred in parallel with a decrease of the affinity of essential SH-groups of cysteine for DTNB in 50% of the oligomeric molecule subunits. Thus, in the dimer two essential SH-groups were rapidly modified by DTNB at the same rate: k1 = k2 = (23.9 +/- 5.6).10(4) M-1 min-1. Within the hexamer, the rate of modification of 3 out of 6 SH-groups was practically unchanged: k1 = (10.6 +/- 2.3).10(4) M-1 min-1. Another 3 SH-groups in the remaining 50% of the subunits were partly masked, which manifested itself in a 10-fold decrease of their modification rate: k2 = (1.12 +/- 0.28).10(4) M-1 min-1. Within the octamer, the SH-groups rapidly interacted with DTNB only on 4 subunits: k1 = (20.7 +/- 2.2).10(4) M-1 min-1, whereas in the remaining 4 octamer subunits a practically complete masking of essential SH-groups was observed, as a result of which these groups became inaccessible to DTNB. This manifested itself in a 1000-fold decrease of the rate of SH-group modification by DTNB which reached that of non-essential SH-group modification. In has been found that a complete loss of the octamer activity is due to the modification of only 4 SH-groups which interact with DTNB at a high rate. A model for subunit association into a dimer, hexamer and octamer has been proposed. Presumably, 50% of the active centers in the mitochondrial creatine kinase octamer are not involved in the catalytic act.

Animals↗
PubMed1986

[Non-equivalency of SH groups essential for the activity of mitochondrial creatine kinase].

The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.

Animals↗
PubMed1985

[Membrane pathology in human myodystrophy].

The authors studied the lipid composition of erythrocytic membranes and levels of pentane (a lipid peroxidation product) in the expired air in patients with Duchenne's myodystrophy. The changes found are discussed in the light of a hypothesis of the generalized membranous defect.

Child↗
PubMed1983

Essential arginine residues of creatine kinase from beef heart mitochondria.

Creatine kinase from beef heart mitochondria is inactivated by 2,3-butanedione. The kinetics of inactivation of the mitochondrial enzyme is biphasic with a bend at a point corresponding to 50% inactivation. The inactivation rate constants of the first fast and the second slow phases of the reaction differ by one order of magnitude, thus suggesting the existence of two types of arginine residues, i.e. "fast" and "slow" ones, with different reactivities. The inactivation rate constant of the slow phase is very close to that for cytoplasmic creatine kinase. At saturating concentrations MgATP and MgADP afford complete protection of the slow phase of inactivation. It is assumed that the "slow" arginine is involved in the binding of metal-nucleotide substrates in the enzyme active center.

Adenosine Triphosphate↗
PubMed1980

[Interaction between mitochondrial creatine kinase and mitochondrial membranes].

The outer mitochondrial membrane of beef heart was disrupted by hypotonic treatment and the effects of concentration and ionic strength of creatine kinase substrate solutions and some other electrolytes and non-electrolytes on mitochondrial membrane creatine kinase were studied. It was shown that electrostatic forces play an important role in the binding of creatine kinase to the mitochondrial membrane. It was assumed that under physiological conditions mitochondrial creatine kinase may undergo a reversible association - dissociation reaction with the membrane. The changes of pH within the range of 6.0 to 9.5 had no effect on the creatine kinase binding to mitochondrial membrane. Creatine kinase from beef heart mitochondria binds likewise and at the same rate to the rat liver no creatine kinase. The number of binding sites in mg of heart mitochondrial protein is n1 = 0.54 +/- 0.11 nmole, Kd1 = 0.16 +/- 0.04 microM. The number of binding sites in mg of liver mitochondrial protein is n2 = 0.65 +/- +/- 0.03 nmole, Kd2 = 0.29 +/- 0.09 microM. Excessive cytochrome c inhibits the binding of creatine kinase to the beef heart mitochondrial membrane. The results obtained suggest that mitochondrial creatine kinase is apparently bound to the membrane phospholipids.

Animals↗
PubMed1980

[Interactions between heart mitochondrial creatine kinase and oxidative phosphorylation].

The conditions for chromatographic separation in Silufol plates of adenine nucleotides, creatinine phosphate, glucose-6-phosphate and Pi have been found. Using this method, it was shown that in the presence of Pi and non-labelled ATP the specific radioactivity of creatine phosphate formed by mitochondrial creatinine kinase via oxidative phosphorylation increases at the same rate as does the specific radioactivity of the surrounding solution of ATP. It is concluded that under the given experimental conditions the ATP formed via oxidative phosphorylation enters the enzyme active center only after it has passed into the solution rather than immediately from the adenine nucleotide carrier.

Adenine Nucleotides↗
PubMed1977

[Study of mechanisms of photoreceptor membrane destabilization under modifying action of oxygen].

Induction of lipid peroxidation in the rod outer segments (ROS) of frog retina results in fragmentation of photoreceptor disc membranes and solubilization of lipoprotein rhodopsin complexes (sedimentation coefficient 2.7S). The substrates of lipid autooxidation are mainly docosahexa- and docosapentaenoyl residues of phosphatidyl ethanolamine. Large fragments (precipitation at 5000 gX30 min) and small vesicles (precipitation at 40 000 gX60 min; average diameter 1000 A) formed from lipoperoxidized ROS differ both in their chemical composition and structural organization. In small vesicles the content of O2-modified polyenoic acyls in the phospholipids is 3,7 times higher as compared to large fragments. Correspondingly, the capacity of hydrophobic areas in the small vesicle membranes evaluated by EPR spin-probing technique is lower than in the large fragments. A mechanism of the photoreceptor membrane destabilization under modification by molecular oxygen is proposed. It is based on a decrease in the value of surface tension upon accumulation of lipid peroxidation products in ROS, the former possessing the properties of non-ionic detergents.

Animals↗