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1.
Anal Biochem ; 309(1): 1-10, 2002 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-12381355

RESUMO

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.


Assuntos
Creatina Quinase/biossíntese , Isoenzimas/biossíntese , Miosinas/biossíntese , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Creatina Quinase/química , Creatina Quinase/isolamento & purificação , Creatina Quinase Forma MM , Deutério , Cromatografia Gasosa-Espectrometria de Massas , Meia-Vida , Isoenzimas/química , Isoenzimas/isolamento & purificação , Cinética , Leucina/análise , Leucina/química , Leucina/genética , Masculino , Peso Molecular , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Miocárdio/química , Miocárdio/enzimologia , Miocárdio/metabolismo , Miosinas/química , Miosinas/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Sequências Repetitivas de Aminoácidos , Tripsina/química
2.
Nat Med ; 5(1): 83-9, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9883844

RESUMO

The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/microl per day and 6 CD8+ T cells/microl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/microl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Infecções por HIV/imunologia , HIV-1/imunologia , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Cinética , Masculino
3.
Anal Biochem ; 267(1): 1-16, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9918649

RESUMO

The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits. Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First, in vitro, using a synthetic peptide present in human serum albumin; and then, in vivo, for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5, 5-2H3]leucine. Additionally, enriched and natural abundance peptides were mixed in vitro to simulate in vivo biosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-2H3]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 +/- 0.03 day-1, t1/2 = 1.53 +/- 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo using proteolytically derived peptides and ESI-MS analysis.


Assuntos
Espectrometria de Massas/métodos , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Deutério , Humanos , Isótopos , Masculino , Oligopeptídeos/síntese química , Oligopeptídeos/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas/síntese química , Proteínas/química , Ratos , Ratos Sprague-Dawley , Albumina Sérica/biossíntese , Albumina Sérica/química
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