Supplemental growth hormone alters body composition, muscle protein metabolism and serum lipids in fit adults: characterization of dose-dependent and response-recovery effects.

Using double-blind, placebo-controlled procedures, the effects of low and high therapeutic dosages of methionyl-human growth hormone (met-hGH) on body composition, muscle protein metabolism and serum lipids were studied in 7 fit adults without growth hormone (GH) deficiency. Dose-dependent changes in body composition were observed that in part appeared to be influenced by a response-recovery effect, as measured by responses factored according to the duration of washout between exposure to the low and high dosages of met-hGH (6 weeks vs. 12 weeks vs. 18 weeks). Increases in fat-free weight were accompanied by an increase in skeletal muscle protein metabolism. Basal levels of cholesterol were inversely related to peak levels of GH in response to exercise stimulation and IGF-I, while GH supplementation lowered levels of total cholesterol and high- and low-density lipoproteins. A dose-dependent effect occurred for total cholesterol, and the percent change in cholesterol was related to the percent change in insulin-like growth factor I (IGF-I). Endogenous levels of GH were attenuated in response to stimulation and IGF-I levels were increased after treatment with GH, but no dose-dependent changes were observed. We conclude that met-hGH alters body composition and muscle protein metabolism, and decreases stored and circulating lipids in fit adults with a pre-existing supranormal body composition. The physiological profile of the person was not as important as the treatment conditions in determining the somatic and physiological response outcomes.

Fetal hyperinsulinemia and protein turnover in fetal rat tissues.

The effects of fetal hyperinsulinemia on protein turnover in various tissues of fetal rats were determined after transuteral injection of insulin to rat fetuses at day 19 of gestation. Tissue protein content was measured on the subsequent days of gestation (days 20-22), and protein synthesis was determined at day 20 of gestation in fetal tissues after intravenous injection of [3H]phenylalanine into the maternal circulation, followed by measurements of tissue free and protein-bound phenylalanine specific radioactivity in fetal diaphragm, brain, heart, and liver. Rates of protein degradation in these fetal tissues were calculated by subtracting protein accretion rates from rates of protein synthesis. The injection of insulin to rat fetuses at day 19 of gestation resulted in relative macrosomia versus saline-injected controls from the same litter (body wt at day 20 of gestation, 3.26 +/- 0.15 g for saline-injected fetuses and 3.60 +/- 0.25 g for insulin-injected fetuses, P less than 0.001) and increased protein and RNA content of brain, heart, and liver. Although fractional rates of protein synthesis were not significantly elevated in tissues from the hyperinsulinemic fetuses, absolute rates of protein synthesis were increased in brain, heart, and liver of hyperinsulinemic fetuses. Hyperinsulinemia did not reduce calculated rates of protein breakdown in fetal brain, heart, or liver but did in fetal diaphragm. We conclude that the major effect of fetal hyperinsulinemia on protein turnover in rats is to increase protein synthesis in selected tissues without simultaneously affecting protein breakdown.

Protein turnover in tissues of the rat fetus following maternal starvation.

The effects of maternal starvation upon protein turnover in various tissues of the fetal rat were determined. Protein synthesis was determined in fetal tissues by the intravenous injection of "massive" amounts of 3H-phenylalanine into the maternal circulation, followed by measurements of tissue free and protein-bound phe specific radioactivity in fetal diaphragm, heart, liver, and brain. Rates of protein degradation in fetal tissues were assessed by subtracting protein accretion rates from protein synthesis. Fractional rates of protein synthesis were minimally affected by maternal starvation in diaphragm, heart, and brain. The major factor contributing to reduced fetal protein accretion in these fetal tissues during maternal starvation was enhanced protein breakdown. These findings differ from those previously reported in young adult rodents following fasting or malnutrition.

The role of spermine in preventing misacylation by phenylalanyl-tRNA synthetase.

Physiological concentrations of polyamines contribute significantly to both the speed and precision of aminoacylation of tRNA. Unphysiologically high concentrations of magnesium ion are required to obtain high rates of synthesis of phenylalanyl-tRNAPheyeast catalyzed by yeast phenylalanine:tRNA ligase in vitro. Under such conditions, rates of misacylation (e.g. the synthesis of Phe-tRNAValE.coli) may be one-fifth of the rate of correct acylation. High rates of correct aminoacylation are achieved in the presence of physiological concentrations of magnesium (1.0 mM) plus spermine (0.2 mM). Under these conditions, there is almost no misacylation. A kinetic study of the magnesium dependence of aminoacylation shows that the rate-determining transition state for Phe-tRNAPhe synthesis contains, in addition to tightly bound Mg2+, either two spermines (Kd = approximately 50 microM) or 2 Mg2+ ions (Kd = approximately 1.0 mM). We postulate that the tRNAPhe binds two spermines to form a very compact, very precisely defined structure that easily forms an activated E.S conformer, E.S (Jencks' Circe Effect). In the absence of spermine, 2 Mg2+ ions bind somewhat more poorly to the tRNA to form a similar but not identical tRNA conformer which, in turn, is less able to form E.S and thus more slowly aminoacylated. Misacylation of noncognate tRNA requires several additional, more loosely bound Mg2+ ions that serve to relax the tRNA structure. Such magnesium-driven relaxation has a minor effect on Km (tRNA), but the resulting floppy structure is able to avoid the barriers against reaction and is amino-acylated thousands of times more rapidly than the compact defined structure.

Conformational changes during enzyme catalysis: role of water in the transition state.

The entropy of activation for the synthesis of Ile-tRNA is high and positive. The only likely source of a high delta S is the loss of structured water as the enzyme . substrate complex moves toward the transition state. This requires a change in the orientation or nature of water-organizing residues in the interface between the enzyme . substrate complex and the water. Such changes, which may be some distance from the "active site," are coupled to the active site in such a way that the increased entropy and decreased free energy of the water--enzyme interface is available at the "active site" to reduce the free energy of activation. The effects of Hofmeister anions on KmS and KcatS are consistent with the entropy data.

The mechanism of the aminoacylation of transfer ribonucleic acid. The kinetics and stoichiometry of the lysis of aminoacyl-tRNA.

It is often stated that the aminoacylation of transfer RNA proceeds in discrete steps: (formula: see article). If this is a complete description of the reaction, the reverse overall formation of ATP should not be more rapid than the formation of Enz . (AA approximately AMP). We show for four different amino acid:tRNA ligases that lysis of AA-tRNA (with PPi and AMP) to ATP is faster than lysis of AA-tRNA (with AMP only) to Enz . (AA approximately AMP). This requires that the transition state proceeds from a quaternary complex of PPi, AMP, AA-tRNA and Enz. From the law of microscopic reversibility, this requires that in the forward reaction the AA-tRNA bond be formed before PPi leaves the enzyme complex. Therefore, the forward reaction passes through the quaternary complex Enz . ATP . AA . tRNA. (In view of recent evidence of the specific requirement of two cations, the complex is accurately described as senary).

The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting.

It has been proposed that the rate-limiting step in the synthesis of aminoacyl-tRNA is the rate at which the product dissociates from the enzyme. The experimental evidence supporting this hypothesis comes from work at low pH and low temperature (although the reaction has been argued to have the same mechanism under physiological conditions). We have reexamined the binding assay by which M. Yarus and P. Berg (1969) (J. Mol. Biol. 42, 171-189) measured the kd for dissociation of Enz-(Ile-tRNA). We find that when overall reaction and dissociation are measured under identical conditions the two rates are not the same. Moreover, while an increase in ionic strength greatly stimulates dissociation, the same increased ionic strength slows aminoacylation. Spermine accelerates overall aminoacylation without affecting dissociation. Because any change in a rate-limiting step must, by definition, cause a parallel change in the overall reaction, these observations prove that under these conditions the synthesis of Ile-tRNA is not limited by the rate of dissociation of Enz-(Ile-tRNA). Entirely similar observations were made for the dissociation of Enz-(Val-tRNA) and the overall synthesis of Val-tRNA at 0 degrees C, PH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacylation was shown not to be saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged substrate tRNA and with product Val-tRNA. E. W. Eldred and P. R. Schimmel ((1972) Biochemistry 11, 17-23) report that the formation of Ile-tRNA proceeds at two rates: (a) k = 2 X 10(-2)S(-1) until the enzyme is saturated with the first mole of product, and (b) k = 2 X 10(-3)S(-1) for subsequent cycles. We did not observe this behavior at any pH or temperature with four different amino acid:tRNA ligases. Because aminoacylation proceeds more rapidly than "dissociation" under some conditions, we believe that the binding assay measures not only enzyme-product dissociation but also other slower reactions such as aggregation or disaggregation of Enz-(AA-tRNA). In conjunction with recent studies from other laboratories, this work makes it unlikely that enzyme-product dissociation is the rate-limiting step in the synthesis of aminoacyl-tRNA either at low temperature and pH or under more nearly physiological conditions. From the effect of salt, it would appear that the rate of aminoacylation of tRNA is largely limited by the rate or extent of formation of Enz-(tRNA) (Loftfield, R. B., and Eigner, E. A. (1967), J. Biol. Chem. 242, 5355-5359). Using the binding assay of M. Yarus ((1972) Biochemistry 11, 2050-2060), we find the Kass for Enz-(Ile-tRNA) varies linearly with the Debye-Hückel function at ionic strengths of 0.1-0.4 from 10(8) to 10(6).

The mechanism of aminoacylation of transfer ribonucleic acid. Reactivity of enzyme-bound isoleucyl adenylate.

Isoleucyl adenylate bound to isoleucine:tRNA ligase of Escherichia coli (EC 6.1.1.5; isoleucyl-tRNA synthetase) transfers the isoleucine moiety to tRNA-Ile-E. coli with a half-time of about 35 s at 0 degrees and pH 7.6 in the presence of spermine or Mg2+. If a limited amount of tRNA-Ile is supplied to a mixture of free enzyme and enzyme-bound [14c]isoleucyl adenylate in a medium containing spermine, ATP, and [3H]isoleucine, almost none of the resultant isoleucyl tRNA is derived from preformed enzyme-bound [14C]isoleucyl adenylate. Almost all of the isoleucyl tRNA formed results directly from reaction of free enzyme, ATP, and isoleucine with tRNA. Similar but less clearcut results are obtained when Mg2+ is substituted for spermine. We conclude that isoleucyl adenylate bound to isoleucine:tRNA ligase is not a significant intermediate in the synthesis of isoleucyl tRNA under these conditions.

Molecular order of participation of inhibitors (or activators) in biological systems.

From a Hill plot it is frequently possible to determine whether exactly one, two, or several inhibitor (or activator) molecules are involved in a critical rate-determining biological process.