[Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000].

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of this enzyme. Methoxy-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of pegylation procedure allowed achieving high level of the enzyme modification. Under 4.5 molar excess of modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for asparaginase production with improved therapeutic properties.

[Identification of AGR2 protein, a novel potential cancer marker, using proteomics technologies].

A comparative analysis of the proteins in prostate tissues of the patients operated for hyperplasia (n = 7) or cancer (n = 5) was performed aiming to search for protein diagnostic markers. Differences in several minor proteins were detected using two-dimensional electrophoresis according to O'Farrel; among them, an additional protein with a molecular weight of 19 kDa and an isoelectric point of 9.0 was observed in four of the cancer cases. Mass spectrometry allowed this protein to be identified as the androgen-induced secreted protein AGR2. The possibility of using AGR2 as a diagnostic marker of prostate cancer is discussed.

[The effect of magnesium pool isotopy on reactivation of mitochondrial ATP synthesis suppressed by 1-methyl-nicotine amide].

The ATP-generating activity of both rat myocardial mitochondria and intramitochondrial creatine phosphokinase (CPK) was examined as a function of the incubation medium magnesium pool isotopy. The in vitro systems tested were prepared from the hearts of animals treated with single injection of 1-methyl-nicotine amide (MNA) suppressing the NAD(P)-dependent reactions in vivo. The presense of the 25Mg paramagnetic cations leads to essential compensation of intramitochondrial ATP deficiency caused by the MNA induced blockade of oxidative phosphorylation. This effect is merely unreachable in those systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The reactivation of mitochondrial ATP synthesis described here involves CPK activity which does not depends on MNA. In this case, a high efficiency of this reactivation seems to be a spin selective phenomenon which requires, predominantly, 25Mg2+ cations.

[New approaches to molecular diagnosis of prostatic cancer].

We compared prostatic proteins in patients operated for adenoma or cancer. 630 protein fractions were obtained from each tissue sample after fractionation by two-dimentional electrophoresis according to O'Farrell. Comparison of the samples from adenoma and cancer showed their difference by 7 proteins among which were isoforms of glyceraldehydes-3-phosphate-dehydrogenase, alpha-collagen and several little known proteins. Most of the cancer patients had the protein with molecular mass 19 kDa and isoelectric point 9.0. By the results of mass-spectrometry this protein was identified as androgen-induced secreted protein AGR2. This protein is considered a potential oncomarker. Prospects of some postgenome technologies for detection of new diagnostic markers of prostatic cancer are discussed.

[Stable isotopes of Mg2+ as activators of the suppressed ATP-generating function of mitochondria].

The ATP-generating activity of rat myocardial mitochondria and intramitochondrial creatine kinase was examined as a function of the isotopy of the incubation medium magnesium pool. The study was performed using in vitro systems prepared from the hearts of animals injected with 1-methylnicotine amide, which suppresses the NAD (NADP)-dependent reactions in vivo. It was shown that the presence of the 25Mg paramagnetic cations essential by compensates for the intramitochondrial ATP deficiency caused by the 1-methyl-nicotine amide-induced blockade of oxidative phosphorylation. This effect is hardey achievable in systems where the magnesium pool consists of isotopes with a zero nuclear spin (24Mg, 26Mg). The restoration of mitochondrial ATP synthesis involves the participation of creatine kinase since the activity of the latter does not depend on 1-methyl-nicotine amide. In this case, the high efficiency of this restaration seems to be a spin-selective phenomenon which requires predominantly 25Mg2+ cations. A possible meaning of the data for further studies on the mechanisms of enzymatic catalysis regulation is discussed.

[ATP-generating creatine kinase: a new compound isolated from Viperidae venom]. / ATP-generiruiushchaia kreatinakinaza: vpervye obnaruzhennyi komponent iada Viperidae.

This is a first report on the presence of creatine kinase, ATP-generating enzyme, in the venom of Vipera xanthia representing the Viperidae family of poisoning snakes. Kinetic and catalytic properties of 40 kD active monomer has been described with a following discussion on the issue of potential of the data listed for further research in either toxic coagulopathy molecular mode or enzyme application dealing with "thrombin-thrombin receptor" interaction studies. An enzyme purification procedure includes hydrophobic (phenyl-Sepharose), anion exchange (MonoQ) and affinity (ADP-Sepharose) chromatographic steps.

Role of matrix metalloproteinases and their inhibitors in tumor invasion and metastasis.

The role of various matrix metalloproteinases (MMP)--such as gelatinases, stromelysins, matrilysin, collagenase-3, and membrane-bound MMP (MB-MMP)--in tumor invasion and metastasis is discussed. Data suggesting significance for malignant growth of the expression level of these enzymes and also of their activators and inhibitors are presented. It is concluded that at different stages of tumor progression the activity of different MMPs is displayed, which is regulated by various growth factors and oncogenes. Different malignancies are characterized by changes in activities of specific MMPs. Data are presented which show significance of the ratio between the MMP activity and that of tissue inhibitors of metalloproteinases (TIMP) in tumor invasion and metastasis, especially in connection with a dual role of TIMP as both MMP inhibitors and activators.

[Gelatinase activity of protein fractions obtained by electrophoresis in polyacrylamide gel]. / Izuchenie zhelatinaznoi aktivnosti v belkovykh fraktsiiakh, poluchennykh élektroforezom v poliakrilamidnom gele.

The modification of a method of determination gelatinolytic activity in protein fractions obtained by one and two-dimensional polyacrylamide gel electrophoresis is presented. The presence of a fraction 115-125 kD among human proteins of cortex matter and glomerulus of kidney and also in myocardium having gelatinolytic activity is demonstrated.

Matrix metalloproteinases of normal human tissues.

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.

Role of matrix metalloproteinases in development of diabetic nephropathy.

This review considers molecular mechanisms that underlie disorders in the structure and metabolism of renal extracellular matrix in diabetic nephropathy. The contribution of the increased synthesis of renal extracellular matrix proteins in the accumulation of renal mesangial matrix is considered, and the important role of the degradation system of the extracellular matrix proteins in the development of fibrosis is also shown. Data on changes in mRNA expression for the matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in various forms of diabetic nephropathy are presented. A correlation is established between changes in the balance of MMP proteolytic activity and TIMP activity and the accumulation of extracellular matrix.

Standardized and extended catalog of major proteins of the human kidney.

A new version of a two-dimensional electrophoretic catalog of proteins of the human kidney and of various morphological and functional structures of the human kidney is presented; it contains information about 179 polypeptides. The following proteins were identified: crystallin, albumin, mitochondrial superoxide dismutase, actin, fatty acid-binding protein, alpha-ATP-synthase, and transferrin. Some protein groups are specific for certain morphological structures.

[Basal membrane proteins]. / Belki bazal'nykh membran.

The review summarizes recent achievements in biochemistry of basal lamina which is highly specialized structural element of extracellular matrix. Structural and functional characteristics of main basal lamina proteins are reviewed including collagen type IV, laminin, nidogen, and heparan sulfate-containing proteoglycans. Special attention is paid to characteristics of structure and biochemical composition of renal glomerular basal lamina which is one of the main components of renal filtration barrier. Possible methods of investigation and characterization of new basal lamina proteins are discussed which help in understanding of biochemical composition of this structure.

[Mapping proteins from various structural regions of human kidney by two-dimensional electrophoresis]. / Kartirovanie belkov razlichnykh strukturnykh otdelov pochki cheloveka metodom dvumernogo élektroforeza.

The protein composition of various structural divisions of human kidney was studied using two-dimensional electrophoresis. Two-dimensional electrophoregrams of the cortical substance of human kidney revealed 165 polypeptide fractions within the pH range of 4.5-7.5, having molecular masses of 10 to 330 kDa. Electrophoresis of glomerular proteins gave 155 fractions with M(r) = 15-300 kDa, whereas fractionation of glomerular basement membrane proteins gave 40 fractions with M(r) = 30-330 kDa within the same range of pH. The M(r) values for all fractions and the relative electrophoretic mobility in the forward direction were determined. A comparative analysis of the electrophoregrams was conducted. The data obtained were used to construct two-dimensional maps of the cortical substance and glomerular proteins of human kidney.

[Electrophoretic analysis of rat kidney glomerulus basal membrane]. / Elektroforeticheskii analiz belkov bazal'noi membrany klubochkov pochek krys.

A technique was developed for analysis of basal membrane proteins from rat kidney glomerulus using two-dimensional electrophoresis by O'Farrell. Basal membranes were isolated from the glomerulus by means of detergent treatment. Various procedures of basal membrane solubilization were studied. About 25 protein fractions with molecular mass from 25 kDa to 330 kDa were detected in the basal membrane after single-dimensional electrophoresis in presence of sodium dodecyl-sulfate. Two-dimensional electrophoresis of the basal membrane proteins and staining of gels with silver nitrate enabled to detect approximately 50 polypeptide fractions with molecular mass from 25 kDa up to 250 kDa at pH 5-7.

The role of isoacceptor transfer RNAs in regulation of age-related changes in the rate of protein synthesis.

In cell-free protein-synthesizing systems containing an S30 extract from liver and brain cortex tissues of 22-day-old fetuses and of male WAG rats (1-900 days old), the minimal rate of protein synthesis was observed in the fetuses, while the maximal one - in 7-day-old animals. The difference in the rates of protein synthesis correlated with the minimal concentration of total tRNA in the former group and with its maximal concentration in the latter. In fetal tissues, an addition to cell-free systems of total tRNA isolated from homologous tissues of 7-day-old animals augmented protein synthesis up to a level observed in 7-day-old animals, whereas in the tissues of animals belonging to other age groups total tRNA had a far less pronounced stimulating effect which decreased with age. Fractionation of total tRNA and analysis of effects of individual tRNAs on protein synthesis demonstrated that the stimulating influence was induced by tRNA(2Arg), tRNA(4Arg) and tRNA(2Val) from brain cortex and by tRNA(2Leu), tRNA(5Leu), tRNA(2Val), tRNA(1Met) and tRNA(2Met) from liver.

Age-related changes in the rates of polypeptide chain elongation.

The time of an average polypeptide chain synthesis, ts, in the liver and brain cortex of rats of various age--from the 17th day of prenatal life up to the 24th month of the postnatal period--was estimated. At the end of the prenatal period the value of t is much higher than in postnatal life. In newborns, the t value is minimal, showing a gradual increase during the postnatal development. Determination of an average molecular mass of newly synthesized polypeptides demonstrated that the increase of t in postnatal life is due to the decrease of the rate of polypeptide chain elongation.

Effect of Zajdela ascites hepatoma on the activity and synthesis of liver histidase of tumor-bearing rats.

The synthesis of histidase occurs only in free polyribosomes. The relative content of histidase synthesizing polyribosomes in rat liver, in Zajdela ascites hepatoma cells and in the liver of tumor-bearing rats is equal to 1.35%, 0.11% and 0.57%, respectively (of the total amount of free polyribosomes). It was found that hepatoma cell sap has an inhibitory effect on the synthesis of proteins in the cell-free system reconstructed from polyribosomes and cell sap of control rats.

[A simple enzymatic method for the quantitative determination of cordycepin and [3'-deoxy]ATP in rat brain tissue]. / Prostoi énzimaticheskii metod kolichestvennogo opredeleniia korditsepina i [3'-dezoksi] ATF v tkani golovnogo mozga krys.

A procedure for quantitative estimation of cordycepin (3'-deoxyadenosine), a specific inhibitor of post-transcriptional polyadenylation of pre-mRNA and of a metabolite of cordycepin 3'-deoxyATP in rat brain tissue is described. The procedure is based on evaluation of an inhibitory effect on the synthesis of polyA in vitro of total nucleoside and nucleoside and nucleoside triphosphate preparations isolated from the animal brain after cordycepin administration. Homogenates of intact animal brain were used as a source of "crude" polyA polymerase preparations.