PROTEIN KINETICS DETERMINED IN-VIVO WITH A MULTIPLE-TRACER, SINGLE-SAMPLE PROTOCOL - APPLICATION TO LACTASE SYNTHESIS

Precise analysis of the kinetics of protein/enzyme turnover in vivo ha s been hampered by the need to obtain multiple tissue samples at diffe rent times during the course of a continuous tracer infusion. We hypot hesized that the problem could be overcome by using an overlapping (i. e., staggered) infusion of multiple stable amino acid isotopomers, whi ch would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning ove r) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overl apping infusion of [H-2(3)]leucine, [C-13(1)]leucine, [C-13(1)]phenyla lanine, [H-2(5)]phenylalanine, [C-13(6)]-phenylalanine, and [H-2(8)]ph enylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-t o-tracee ratios of each isotopomer were measured in the plasma and jej unal free amino acid pools as well as in purified LPH polypeptides. Th ese values were used to estimate kinetic parameters in vive using a li near steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 +/- 1.1%/min, 17.4 +/- 11 %/min, and 0.089 +/- 0.02 %/min, respectively. We conclude that this multipl e-tracer, single-sample protocol is a practicable approach to the in v ive measurement of protein fractional synthesis rates when only a sing le tissue sample can be obtained. This method has broad application an d should be particularly useful for studies in humans.