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Reaction of phenylglyoxal with chicken gizzard myosin
Abstract:
Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.
Insights
Phenylglyoxal modification of chicken gizzard myosin inhibits K+-ATPase activity by targeting arginine residues. This modification, particularly in the subfragment 1 region, alters protein conformation, reducing enzymatic function and highlighting arginine
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Chicken gizzard myosin is a key contractile protein.
- K+-ATPase (ATP phosphohydrolase) activity is crucial for muscle function.
- Understanding myosin modification provides insights into contractile mechanisms.
Purpose of the Study:
- To investigate the effect of phenylglyoxal modification on chicken gizzard myosin's K+-ATPase activity.
- To identify the specific residues and regions of myosin affected by phenylglyoxal.
- To elucidate the role of arginine residues in myosin's ATPase activity.
Main Methods:
- Chemical modification of chicken gizzard myosin using phenyl[2-14C]-glyoxal.
- Assay of K+-ATPase activity over time.
- Spectrophotometric determination of reagent incorporation and residue modification.
- Analysis of myosin fragmentation and light chain integrity.
- Investigation of substrate (MgATP2-, MgADP) effects on modification and activity.
Main Results:
- Phenylglyoxal inhibited K+-ATPase activity in a time-dependent manner, with 50% inhibition at 3.2 mol reagent incorporation per 4.7 X 10(5) g protein.
- Phenylglyoxal reacted with arginine residues in a 2:1 molar ratio (phenylglyoxal:arginine).
- Modification primarily occurred in the subfragment 1 heavy chain and rod regions, without light chain loss.
- Inhibition correlated with modification of the subfragment 1 region.
- Nucleotide substrates (MgATP2-, MgADP) enhanced inactivation and altered reagent distribution.
- Conformational changes induced by modification were responsible for the observed inhibition.
Conclusions:
- Arginine residues are essential for maintaining the ATPase activity of chicken gizzard myosin.
- Modification of specific arginine residues in the subfragment 1 region leads to conformational changes and loss of enzymatic function.
- The findings highlight the critical role of arginine residues in the catalytic activity of myosin.