[Preservation of beta-carotene during production and storage of sterilized milk]. / Sokhrannost' beta-karotina pri proizvodstve i khranenii sterilizovannogo moloka.

Safety of beta-carotene in sterilized dairy products under different methods of sterilization, keeping conditions, packing and food form of carotene has been investigated. The standard documentation on sterilized milk "Provita" with different level of fat and content of beta-carotene (0.25 mg/100 g) has been developed.

[Refining criteria for supplying the body with vitamin B6]. / Utochnenie kriteriev obespechennosti organizma vitaminom B6.

As shown by many-year experience of human vitaminization with vitamin B6 and by mathematical analysis of a relationship between content of pyridoxal phosphate in blood plasma, excretion of 4-pyridoxic acid with urine and the rate of PALP-effect as well as after calculation and mathematical interpretation of statistical variations in distribution of those pyridoxal phosphate content in blood plasma and the rate of PALP-effect in man after additional vitaminization, the value of PALP-effect not exceeding 2.5 and content of PALP more than 8ng/ml in blood plasma should be recommended as a criterion of normal vitamin B6 consumption. Excretion of 4-pyridoxic acid with urine more than 60mg/h in children 5-7 years old and 70 mg/h in adults and children 9 years appear to serve as a criterion of normal vitamin B6 consumption.

[Dietary plant phospholipids as stabilizers of oil solutions of beta-carotene]. / Fosfolipidy rastitel'nye pishchevye kak stabilizatory maslianykh rastvorov beta-karotina.

A study of stability of oil solutions beta-carotene to oxidizing damage is carried out. As stabilizers were used dietary plant phospholipids produced by Krasnodar's company ECOTECH in concentrations 5, 10, 20% in a combination with alpha-tocopherol. Oxidizing changes in samples were evaluated by determination of peroxide level ('peroxidation number') and contents diene conjugates. Effect of these antioxidants was studied in experiments with accelerated oxidation at 60 C in darkness during 15 days. Phospholipids entered in a sample acted as antioxidants. Reverse correlation was found between quantity added phospholipids an level of accumulated primary peroxidation products. It was shown that during experimental oxidation together with oxidizing damage of oil the loss of beta-carotene was also occurred: without antioxidants on 68%, with alpha-tocopherol alone on 27%, with phospholipids and alpha-tocopherol on 34%. Phospholipids protect an oil from oxidizing damage not rendering of direct action on safety (-carotene being only as synergists in a combination with alpha-tocopherol. Phospholipids increase thus antioxidant potentiation of a product lying as consider in the basis of antioxidant action of beta-carotene.

[Refining criteria for providing the body with vitamin B2]. / Utochnenie kriteriev obespechennosti organizma vitaminom B2.

The following patterns should be recommended as a criterion of normal vitamin B2 requirements: the FAD-effect is no more than 1.25, the levels of riboflavin, more than 5 ng/ml in the plasma and more than 130 ng/ml in the erythrocytes. At the same time, the hourly urinary riboflavin excretion more than 10 g/h may be used as a criterion of normal vitamin B2 requirements in children of 5-7 years old. These values were obtained while analysing the many-year experimental data on riboflavin treatment and using the mathematical analysis of the dependence curves for the urinary riboflavin excretion, its levels in plasma and erythrocytes, and the value of the FAD effect, as well as deriving and mathematically interpreting the variation curvers for the distribution of the given value of the FAD effect and the plasma riboflavin levels for human beings after additional vitamin therapy.

[Isolation of riboflavin-binding apoprotein from chicken egg protein and its use for determining riboflavin in biological samples]. / Vydelenie riboflavinsviazyvaiushchego apobelka iz belka kurinykh iats i ego ispol'zovanie dlia opredeleniia riboflavina v biologicheskikh obraztsakh.

A riboflavine-binding protein has been isolated from egg white and its properties have been characterized. The apoprotein-employing techniques for riboflavine detection in foods, urine, and blood serum are highly competitive with conventional labor-consuming procedures and with the high expensive HPLC method in sensitivity and selectivity, and can be recommended for riboflavine detection in biological objects.

[The choice of adequate criteria for the vitamin B6 allowance of the human body]. / K vyboru adekvatnykh kriterii obespechennsoti organizma cheloveka vitaminom B6.

Correlations between indicators of vitamin B6 supply of the body were studied: blood plasma pyridoxal phosphate (PP) level, red cell basal specific activity of aspartate aminotransferase (AsAT), PP effect, urinary excretion of 4-pyridoxilic acid (4-PA). In normal riboflavin supply of the body all the four indicators are in good correlation and may be regarded as criteria of vitamin B6 supply. Blood plasma PP concentration over 8.5 ng/ml, urinary excretion of 4-PA more than 60 micrograms/h should be used as the lower thresholds of the normal range in high-pressure liquid chromatography. Red cell AsAT activity and the magnitude of PP effect depending on specific conditions (storage, hemolysate dilution, substrate concentration, etc.), their use is permissible only after standardization of these conditions and setting up the values conforming to the norm for the specific conditions of AsAT activity measurements. Under conditions of our studies PP effect magnitude more than 2 points to vitamin B6 deficit.

[The binding of SSB-proteins with DNA in chromatin of Ehrlich ascites carcinoma cells]. / Sviaz' SSB-belkov s DNK v khromatine kletok astsitnoi kartsinomy érlikha.

Using UV-induced cross-linking between proteins and DNA, the contacts between single-stranded DNA-binding proteins (SSB proteins) and chromatin DNA have been demonstrated. Ehrlich ascites tumour DNA was labeled in vivo by inoculation of tumour-bearing mice with 3H-thymidine. The cells were irradiated with the UV light dose of 3000 J/m2, destroyed in a Triton X-100-containing hypotonic medium, and separated by centrifugation into the extrachromatin fraction and chromatin. Chromatin DNA was digested with DNAase 1, and the chromatin proteins were extracted with 2 M NaCl-polyethyleneglycol. SSB proteins from the extrachromatin fraction and chromatin were purified. Only SSB proteins from UV-irradiated cell chromatin appeared to possess a high specific radioactivity which exceeded 7.5-fold that of non-irradiated cells. There were no differences between chromatin SSB proteins in control and irradiated cells as could be evidenced from SDS electrophoresis data. It is assumed that in irradiated cells SSB proteins of DNA-digested chromatin are covalently cross-linked with DNA fragments.

Properties of single-stranded DNA-binding proteins (SSB-proteins) from chromatin and nonchromatin fractions of Ehrlich ascites tumour: phosphorylation enhances the affinity of SSB-proteins for single-stranded DNA.

To assess the possible functional role of single-strand DNA-binding (SSB) proteins in eucaryotic cell, a comparative study was made of SSB-proteins isolated from chromatin and the nonchromatin fractions of Ehrlich ascites tumour (EAT) cells. No appreciable differences between the two groups could be found either in SDS-gel electrophoretic patterns or in the ssDNA-binding capacity and stimulation of DNA replication in permeable EAT cells. However, the chromatin SSB-proteins incorporated 1.4-times more labelled phosphate in vivo; phosphate assays in the isolated chromatin and nonchromatin SSB-proteins yielded ca. 3 and 2 moles Pi/mole protein, respectively. Both preparations could be further phosphorylated in vitro with Ca-phospholipid-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase, but the non-chromatin proteins were phosphorylated to a greater degree. In parallel with phosphorylation, the SSB-proteins displayed stronger binding to ssDNA cellulose. Phosphorylation may thus be a means of regulating the functions of SSB-proteins, in particular their interaction with chromatin.

[A comparison of fluorescent methods of determining the concentration of vitamin B2 in blood]. / Sravnenie fliuorestsentnykh metodov opredeleniia kontsentratsii vitamina B2 v krovi.

Three methods of riboflavin assay in the human blood were comparatively studied. It has been found that firmly-bound flavin forms present in erythrocytes inhibit their complete transmission into the riboflavin form. Low specificity of the method using riboflavin fluorescence suppression with dithionite has been proved. Dithionite capacity for suppressing fluorescence of riboflavin-like substances significantly rises detectable levels. The use of riboflavin-binding protein permits one to determine only free riboflavin form. It has been established that the lumiflavin method is most reliable and specific for riboflavin assay in erythrocytes.

[Phosphorylation and other properties of proteins binding single-stranded DNA (SSB-proteins) from chromatin and extrachromatin fractions of Ehrlich ascites carcinoma]. / Fosforilirovanie i drugie svoistva belkov, sviazyvaiushchikhsia s odnonitevoi DNK (SSB-belkov) iz khromatina i vnekhromatinovoi fraktsii kletok astsitnoi kartsinomy Erlikha.

A comparative study of single-stranded DNA-binding proteins (SSB-proteins) isolated from chromatin and the extrachromatin fraction of Ehrlich ascites tumour cells was carried out. No differences were found either in SDS-gel electrophoretic mobility or in the single-stranded DNA-binding capacity and stimulation of the replicative synthesis of DNA. However, chromatin SSB-proteins contained 1.4-1.5 times more phosphate than extrachromatin proteins. Both preparations could be phosphorylated in vitro by protein kinase C and cAMP-dependent protein kinase, but the chromatin proteins were phosphorylated in a lesser degree. In parallel with phosphorylation the SSB-proteins displayed a higher binding affinity for ssDNA-cellulose. Phosphorylation can thus be regarded as a means of regulation of the SSB-protein function, in particular, their interaction with chromatin DNA.

[Phosphorylation of isolated components of the troponin complex of skeletal and cardiac muscle phosphorylase kinase from bird skeletal muscles]. / Fosforilirovanie izolirovannykh komponentov troponinovogo kompleksa skeletnoi i serdechnoi myshts kinazoi fosforilazy iz skeletnykh myshts ptits.

Pigeon and chicken skeletal muscle phosphorylase kinase purified to a nearly homogeneous state is able to phosphorylate both cardiac and skeletal troponin I and T. After 1-hr incubation, the enzyme transfers up to 0.35 mole of phosphorus per mole of skeletal troponin I, up to 0.5 mole of cardiac troponin I and up to 0.1 mole of cardiac and skeletal troponin T. Avian muscle phosphorylase kinase does not phosphorylate the first serine residue of cardiac and skeletal troponin T, but catalyzes the phosphate incorporation into the site(s) of troponin T located in the central or C-terminal parts of the protein molecule. The rate of troponin phosphorylation by pigeon muscle phosphorylase kinase is pH-dependent: the 6.8/8.2 ratio for troponin I is close to 0,2, whereas that with troponin T varies in the range of 0.5-0.7. Troponin phosphorylation by avian phosphorylase kinase depends on the presence of Ca2+ in the incubation mixture. In the presence of 3 mM EGTA troponin I phosphorylation is inhibited by 70-90%, whereas that of troponin T--by 50%. The experimental results indicate that the phosphorylation of troponin I and T is catalyzed either by two different active centers or by different conformations of the single center of avian phosphorylase kinase.

Casein kinases I and II bound to pig brain microtubules.

1. Microtubules prepared from pig brain by two cycles of assembly-disassembly comprise cyclic nucleotide-independent protein kinase activity with phosvitin and troponin T as substrates. 2. Phosphocellulose chromatography resolved two phosvitin kinase activity peaks, one of which coincided with the troponin T kinase peak. 3. The activity peak corresponding to troponin T kinase was inhibited by heparin (I50 = 0.06 micrograms/ml), whereas the other phosvitin kinase peak was unaffected. 4. Both kinase fractions phosphorylated tubulin and microtubule-associated protein (MAP-2). 5. It is concluded that pig brain microtubules contain bound casein kinases I and II. The association may target the action of these kinases toward microtubular proteins in vivo.

[Phosphorylation of troponin in the heart and skeletal muscle by Ca2+-phospholipid-dependent protein kinase]. / Fosforilirovanie troponina serdtsa i skeletnykh myshts Ca2+-fosfolipidzavisimoi proteinkinazoi.

The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo.

Phosphorylation of troponin T by Ca-phospholipid-dependent protein kinase.

Phosphorylation of skeletal and cardiac troponin T by Ca-phospholipid-dependent protein kinase/protein kinase C/was investigated. Under conditions used, the rate of troponin T phosphorylation was only 3-4 times lower than that of histone H-1, and after long term incubation the enzyme incorporated about 1 mol of phosphate per mol of skeletal and cardiac troponin T. The sites phosphorylated by protein kinase C are mainly located in the C-terminal part/residues 159-259/of skeletal troponin T. The less effectively phosphorylated sites are located in between residues 42-158 of troponin T. Reverse phase HPLC revealed four groups of tryptic phosphopeptides of troponin T. The phosphopeptides weakly adsorbed on a RP-18 column were identified as a mixture of two peptides/GKKQTAR and QTAR/both containing Thr-171 of troponin T phosphorylated by protein kinase C.

[Type-II casein kinases: characteristics of their properties and possible physiological role]. / Kazeinkinazy II tipa: kharakteristika svoistv i vozmozhnaia fiziologicheskaia rol'.

A detailed characteristic of casein kinases of the second type based on the analysis of the literary data and own research is given. The main properties which allow to differ them from other protein kinases are described. The participation of casein kinases II in regulation of different cellular processes are shown: in carbohydrate and lipid metabolism, in transcription, translation and enzyme synthesis. The changes in the activity of the given casein kinases in the course of the cellular cycle and during some pathological states show that from the diagnostic point of view the measurements of the enzyme activity is a useful tool, especially in an early detection of uncontrolled cell proliferation. On the basis of the given data the casein kinases II have been concluded to play an important role in the control of organism normal development. The given proteins may be put in one line with well studied and physiologically important cAMP-dependent and Ca-dependent protein kinases.

Comparison of the structure of two cardiac troponin T isoforms.

Two isoforms of troponin T have been isolated from bovine cardiac muscle. One isoform has an Mr of 31000 and a pI at about 7.1, the corresponding values for the second isoform being 33000 and 6.5. Both isoforms have identical C- and N-terminal sequences, and, according to the data from tryptic-peptide mapping, a similar structure of the central and C-terminal domains. The large N-terminal peptides of troponin T isoforms differ in the content of glutamine/glutamic acid and alanine. It is concluded that the isoform with Mr 33000 has an additional peptide enriched with glutamic acid and alanine that is inserted between the N-terminal pentapeptide and the cysteine located 40-60 residues from the N-terminus.

Some properties of the nucleotide-binding site of troponin T kinase-casein kinase type II from skeletal muscle.

Investigation of properties of skeletal muscle troponin T kinase (EC 2.7.1.37) has revealed that the enzyme belongs to the group of casein kinases of the second type. The enzyme consists of two subunits with apparent molecular weights of 44 000 and 26 000 and contains a protein with molecular weight of 39 000, which is probably the proteolytic fragment of the 44 000 subunit. The substrate specificity of troponin T kinase was tested, using 20 analogs of the nucleotide. The enzyme has a low substrate specificity toward the purine base and uses both ATP and GTP as substrates. Modification of the ribose ring does not influence the enzyme interaction with the nucleotide; however, the cleavage of ribose leads to a decrease of the enzyme-nucleotide interaction. Elimination of the gamma-terminal phosphate or its modification by bulky hydrophobic radicals do not affect this interaction. A comparison of the Ki values for different analogs suggests that the interaction of troponin T kinase with the nucleotide occurs via the binding of the purine base and the beta-phosphate group of the analog.

[Troponin T kinase: possible relationship to casein kinases of the G type]. / Dal'neishee izuchenie svoistv troponin-T-kinazy: vozmozhnaia analogiia s kazeinkinazami G-tipa.

Troponin T kinase utilizes ATP and GTP as substrates for the protein kinase reaction. When phosvitin is used as substrate, the enzyme activity increases with an increase in the ionic strength up to 0.2-0.3 M KCl. The pH optimum for the enzyme lies at 8-9. Ultracentrifugation in sucrose density gradient showed that the sedimentation coefficient of troponin T kinase is 9.5 S. The Stocks radius determined by gel-filtration is equal to 49 A. The molecular weight of the enzyme calculated from the given values of the Stocks radius and sedimentation coefficient is 184,000. This is indicative of the oligomeric structure of the enzyme and suggests that the stoicheiometry of the enzyme subunits having mol. weights of 50,000, 46,000 and 31,000 is other than 1:1:1. Troponin T kinase is capable of autophosphorylation; the phosphorylation process involves only the protein with mol. weight of 31,000. The data obtained suggest that troponin T kinase can be referred to casein kinases of G type and may participate in translation processes.

Phosphorylase kinase phosphorylation of skeletal-muscle troponin T.

Rabbit skeletal-muscle troponin T was phosphorylated by a standard preparation of phosphorylase kinase [Cohen (1973) Eur. J. Biochem. 34, 1--14] and by fractions obtained after chromatography of phosphorylase kinase on phosphocellulose. The original preparation of phosphorylase kinase phosphorylated at least two sites, one of which was serine-1. The second and probably the third sites were presumably located in the peptide flanked by amino-acid residues 147 and 161 of troponin T. Fractions of phosphorylase kinase was adsorbed on phosphocellulose phosphorylated only the second site. Tightly adsorbed fractions possessed high troponin T kinase and phosvitin kinase activities and phosphorylated only serine-1 of troponin T. The results suggest that standard preparations of phosphorylase kinase are contaminated by troponin T kinase, which can phosphorylate serine-1 of troponin T.