We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)
Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(-) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(-) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA0) to HA1 and HA2, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(-) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs.
The biochemical properties of strains (PT-R101 and PT-R108) of Escherichia coli K12 resistant to growth inhibition by pyrithiamine, and antimetabolite of thiamine, have been studied. Intracellular thiamine pyrophosphate concentration in these resistant strains was slightly but definitely higher than that in the parent strain. Thiamine synthesis from the pyrimidine and thiazole moieties of thiamine by cell suspensions was greater in the resistant strains than the parent strain. The activities of enzymes involved in thiamine biosynthesis in these pyrithiamine-resistant strains were 2-3 times higher than the parent strain (3301), except for thiamine-phosphate kinase, which was indetectable in in vitro assay of the activity. However, other evidence indicates that this enzyme is not defective but is functioning in vivo and, furthermore, that the negligible activity of this enzyme did not affect the growth rate of the mutants. The activities of these enzymes were further enhanced when PT-R101 was grown on 5mM adenine and were reduced almost to zero when the strain was grown on 0.1 muM thiamine in the same way as the parent strain. However, when these resistant strains were grown on a low concentration of thiamine such as 0,05 muM, thiamine synthesis by cell suspensions also decreased, but only to a limited extent compared with the parent strain. These results suggest that PT-R101 and PT-R108 are altered in the mechanisms of regulation of thiamine biosynthesis. Their altered properties might be due to a reduced binding affinity of the repressor protein, which is involved on the regulation of thiamine synthesis, for the corepressor, thiamine pyrophosphate,
Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg 2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme.Creatine Cofactor Thiamin-diphosphate kinase Thiamin triphosphate
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