Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Improved Dot-Blot Hybridization Assay for Large-Scale Detection of Potato Viruses in Crude Potato Tuber Extracts

Abstract A rapid and simple technique has been developed to enhance the sensitivity of virus detection in dot-blot hybridization assay by up to 1000 fold. The procedure generally follows that of B aulcombe et al. (1984) but includes moderate heating of the nitrocellulose filter in 10XSSC, 0.5% SDS solution at 55°C after sample application. Using this procedure, four potato viruses (PVX, PVS, PVM and PVY) were detected with cloned virus-specific ³²P-cDNA probes in 2 μl spots containing 0.2–2 pg of purufied virus (0.1–1 ng/ml). The procedure was also successfully applied for the detection of PVX, PVS, PVM and PVY in crude potato tuber extracts.     

Chemico-enzymatic synthesis and cloning of human angiogenin gene in M13mp8 phage

The nucleotide sequence coding for human angiogenin has been deduced from the published amino acid sequence with the use of codons preferentially utilized in highly expressed E. coli genes. It was divided into forty-three oligonucleotides, which were synthesized by automatic gene assembler and then joined by DNA ligase into three double-stranded blocks, the blocks were consequently cloned and ligated in M13mp8 phage, and the resultant 389-bp DNA sequence coding for human angiogenin was analysed by chain-terminator sequencing technique.     

Structural and functional analysis of the respiratory burst in the phagocytosing macrophage

The interrelation between structural changes and oxygen consumption by the phagocyting macrophage was studied. The mean number of phagocyted particles was estimated by the method of stereological transformation. It is found that the uptake of yeast particles and CN- -nonsensitive oxygen consumption is related to the concentration of yeast cells in the incubation medium. A positive correlation was established between the oxygen consumption and the mean number of phagocyted particles. The results obtained may suggest that the "respiration burst" takes place in the contact area of the macrophage and the phagocyted material, and its extent probably depends on the surface of that contact area.     

A-like cells of the gastric mucosa as a possible source of prostaglandins

AL cells of the oxyntic stomach area were studied in rats using ultrastructurometric technique. High-threshold, short-term direct electrical vagostimulation (5 V, 4 msec, 30 Hz, 10 sec) was performed in experimental group of 12 animals. Animals were killed 1, 10 and 30 min after stimulation. High post-stimulation lipolytic activity of AL cells and intensification of "granule autophagy" phenomenon were noted. Our findings as well as the literature data suggest a hypothesis on possible prostaglandin production by AL cells. Direct evidence in favour of this hypothesis is difficult to obtain due to the lack of sufficiently reliable methods of their morphological detection in cells.     

Immunoenzyme detection of specific brain antigens as a criterion of the permeability of the hemato-encephalic barrier in rats following acute gamma irradiation

The immunoenzyme detection systems for the measurement of the alpha-2 globulin of the brain (alpha 2M) and glial fibrillary acidic antigens (GFAP) were developed. These systems were used for the study of the penetration through hemato-encephalic barrier in rats subjected to gamma radiation. This method is recommended for the indirect evaluation of the hemato-encephalic barrier functional disorders.     

Effect of apomorphine on rat brain opiate receptors

Stereospecific binding of apomorphine to rat brain opiate receptors was shown by assaying the competition of 7,8(n)--3H--naloxone and D-ala2-tyrosyl-3,5-3H--enkephalin (5-D-leucine) for opiate receptor binding. EC-NaCl50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone and D-ala2, D-leu5-enkephalin in the absence of NaCl were 20 and 42 microM, respectively. EC+NaCl 50, the concentration of apomorphine which inhibited 50% binding of the radioactive naloxone in the presence of 100 mM NaCl was 17 microM. From the ratio of EC+NaCl 50 to EC-NaCl the value of "sodium shift" of effective concentration can be calculated as 0.85. From the data obtained it is concluded that apomorphine, like naloxone, is a "pure" antagonist but it has much less affinity for enkephalin and opiate binding sites. The probable mechanisms of the pharmacological action of apomorphine are discussed.     

The mechanism of action of tRNA methylases studied with immobilized tRNAs.

Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.