Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry.
We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [(2)H(3)]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M(+1) and M(+2) mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3-10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.
Papageorgopoulos
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C
Department of Nutritional Sciences & Toxicology, 309 Morgan Hall, University of California, Berkeley, CA 94720-3104, USA.
Caldwell
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K
Schweingrubber
H
H
Neese
R A
RA
Shackleton
C H L
CH
Hellerstein
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M
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D052283:Creatine Kinase, MM Form; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006207:Half-Life; D007527:Isoenzymes / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D007930:Leucine / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D008297:Male; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D009218:Myosins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D020449:Repetitive Sequences, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D007527:Isoenzymes; D010446:Peptide Fragments; D012346:RNA, Transfer, Amino Acyl; D003903:Deuterium; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form; D014357:Trypsin; D009218:Myosins; D007930:Leucine