Synthesis and physicochemical properties of protein conjugates with water-soluble poly(alkylene oxides).

Conjugates of proteins (bovine serum albumin (BSA) and alpha-chymotrypsin (CHT) with poly(ethylene glycol) and amphiphilic block copolymers of ethylene oxide and propylene oxide (proxanols) were synthesized, using monoaldehyde polymer derivatives as the amino group modifying reagents. Four types of conjugates varying in the placement of hydrophobic block and type of polymer chain distribution were obtained. Methods of purification and characterization of proteins conjugated with proxanols were developed. It was shown that conjugates based on CHT retain high enzymatic activity toward both substrates investigated--N-benzoyl-L-tyrosine and casein-up to high degrees of modification (11 polymer chains per protein molecule). At the same time, CHT--proxanol conjugates were characterized by higher thermostability, the stabilizing effect increasing in parallel with the degree of modification. It was shown that the alteration of sedimentation coefficients of proteins caused by modification was negligible. On the basis of data obtained by the methods of hydrophobic chromatography, sedimentation, and differential scanning calorimetry, conformational models of protein-proxanol conjugates were suggested. It was supposed that conjugates form compact structures in aqueous solutions, which resemble intramolecular micelles, stabilized by hydrophobic interactions between poly(propylene oxide) blocks of proxanols.

Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride.

The thermal unfolding of the myosin subfragment 1 (S1) in its stable complex with ADP and beryllium fluoride (S1.ADP.BeF3-) was studied by differential scanning calorimetry. It has been shown that the structure of the S1 molecule in the S1.ADP.BeF3- complex is similar to that of S1 in its complex with ADP and orthovanadate (S1.ADP.Vi) but differs radically from that of nucleotide-free S1 and S1 in the S1.ADP complex. It is concluded that the S1.ADP.BeF3- complex can be considered, like the S1.ADP.Vi complex, a stable structural analogue of the myosin head.ADP.Pi transition state of the myosin-catalyzed ATP hydrolysis.

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.

The effect of alkali light chains on the thermal stability of myosin subfragment 1.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.

Domain structure of myosin subfragment-1. Selective denaturation of the 50 kDa segment.

The structure of the myosin subfragment-1 (S1) from rabbit skeletal muscle was studied using differential scanning microcalorimetry. Three independently melting regions (domains) were revealed in S1. Selective denaturation of the middle 50 kDa segment of the S1 heavy chain resulted in the disappearance of the heat sorption peak corresponding to the melting of the first, the most thermolabile domain without any effect on the thermally induced blue shift of the intrinsic tryptophan fluorescence spectrum which occurs within the temperature region of melting of the second domain. It is concluded that the most thermolabile domain seems to correspond to the N-terminal part of the 50 kDa segment devoid of tryptophan residues.

[Metabolic effect of gutimine on the myocardial tissue in blood loss]. / Metabolicheskii éffekt gutimina v tkani miokarda pri krovopotere.

A rate of hemorrhage accounted for 31 mg/kg in 80 adult dogs premedicated with promedol and atropine. After circular-hemic hypoxia caused by the hemorrhage, single intravenous administration of gutimine (35-40 mg/kg) transformed the carbohydrate metabolism in heart muscle: activated glycolysis and accelerated the rate of its products oxidation, maintained the free amino acids concentration at the level similar to that of intact animals, reduced the rate of creatine phosphate synthesis.

[Effect of sodium oxybutyrate on macroergic phosphates, function and ultrastructure of the myocardium in blood loss]. / Vliianie oksibutirata natriia na makroérgicheskie fosfaty, funktsiiu i ul'trastrukturu miokarda pri krovopotere.

Experiments on dogs with long-term hypotony caused by blood loss have demonstrated irreversible lesions in the majority of cardiomyocytes and hypodynamic condition of the heart with a high content of macroergic phosphates in the myocardium. Intravenous injection of sodium hydroxybutyrate in a dose of 180-200 mg/kg after 60 min of hypotony seems to improve energy transport and utilization in the ischemic myocardium. It increases the working capacity and power of the heart muscle, pump function of the heart and makes longer the period of ultrastructural changes in cardiomyocytes.