110 research outputs found
The incorporation of inorganic phosphate by rabbit skeletal muscle
Cheesman and Hilton (1961, 1966) and Cheesman and Whitehead (1969) have demonstrated an uptake of 32P by frog muscle. A reversible 40% reduction in the specific activity of bound P was reported to occur on membrane depolarisation. A similar reduction v/as found by Whitehead (1970) when myofibrils prepared from 32P-labelled rabbitpsoas were contracted. Miihlrad et al. (1963) have shown direct uptake--- of 32P by glycerol-extracted myofibrils and by myosin. The present report attempts to consolidate and expand the above observations. A procedure allowing the resolution of myofibrils into P-containing fractions was developed; several modifications were made to existing methods for P determination. Methods for the direct investigation of myofibrillar ATPase, actomyosin ATPase, adenylate kinase and 5'-AMP deaminase activities in the pH range 7-8 and two methods for the estimation of IMP are presented. A circuit diagram for an integrator of pulses from a radioactivity counter is given. An appendix contains practical details of these methods. The possibility was excluded that ATP synthesised from actin-bound ADP during water extraction of acetone-dried muscle derived its y-phosphate group from a source other than the adenylate kinase reaction proposed by Tsuboi (1963). Changes in concentrations of ATP, ADP, AMP and IMP during the extraction were found to be consistent with adenylate kinase and 5'-AMP deaminase activities. Contaminating bacteria were shown to be responsible for direct uptake of 32P by myofibrils and myosin. The reduction, on contraction, in the specific activity of bound 32P in myofibrils prepared from labelled psoas was related to a decrease in extractability of protein from contracted myofibrils. Label was3distributed throughout the P-containing fractions although the nucleotides showed very low activity. Application of myofibrils to polyacrylamide gels yielded several labelled bands. A new role for methylated amino acid residues in muscle is proposed. The roles of adenylate kinase and 5'-AMP deaminase and a contraction mechanism involving the reversible phosphorylation of actin-bound ADP are considered.<p
Role of the HPRG Component of Striated Muscle AMP Deaminase in the Stability and Cellular Behaviour of the Enzyme
Multiple muscle-specific isoforms of the Zn2+ metalloenzyme AMP deaminase (AMPD) have been identified based on their biochemical and genetic differences. Our previous observations suggested that the metal binding protein histidine-proline-rich glycoprotein (HPRG) participates in the assembly and maintenance of skeletal muscle AMP deaminase (AMPD1) by acting as a zinc chaperone. The evidence of a role of millimolar-strength phosphate in stabilizing the AMPD-HPRG complex of both AMPD1 and cardiac AMP deaminase (AMPD3) is suggestive of a physiological mutual dependence between the two subunit components with regard to the stability of the two isoforms of striated muscle AMPD. The observed influence of the HPRG content on the catalytic behavior of the two enzymes further strengthens this hypothesis. Based on the preferential localization of HPRG at the sarcomeric I-band and on the presence of a Zn2+ binding motif in the N-terminal regions of fast TnT and of the AMPD1 catalytic subunit, we advance the hypothesis that the Zn binding properties of HPRG could promote the association of AMPD1 to the thin filament
AMP Deaminase From Saccharomyces cerevisiae
Attempts were made to clone the yeast adenosine-5'-monophosphate deaminase (AMP deaminase) gene from S. cerevisiae using a number of strategies. Initially, it was hoped that overproduction of AMP deaminase could be detected in yeast, by selecting for resistance to the AMP deaminase inhibitor, deoxycoformycin (dCF). This involved the construction of ade, hpt1 yeast strains in which the AMP deaminase reaction is the major source of inosine-5'-monophosphate (IMP). Although yeast AMP deaminase is sensitive to dCF in vitro no effect was observed in vivo (possibly due to lack of uptake of the drug)
Annotated Cell and Molecular Biology 5e: What We Know and How We Found Out
https://dc.uwm.edu/biosci_facbooks_bergtrom/1013/thumbnail.jp
Basic Cell and Molecular Biology 5e: What We Know and How We Find Out
https://dc.uwm.edu/biosci_facbooks_bergtrom/1014/thumbnail.jp
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