Ratio Of Specific Activity Research Articles

Tritium fractionation in anomalous water bound to environmental samples

The quantification of the hydrogen isotope effect involved in the physi- and chemisorption of water on to environmental samples provides the possibility for the characterization of their bound water. This is realized by distilling suspensions of samples in tritiated water ( HTO H 2O ) under near-equilibrium conditions at 5 °C and 5 mbar, and measuring the specific tritium activity in fractions of the condensed gas phase in the course of water removal. The tritium fractionation is caused by isotope effects during the evaporation of water as well as during the sorption of water on to the sample matrix. The two isotope effects are separated by comparing the fractionation in the sample—water mixtures with that in pure water. Three typical soils and maize plant seeds are chosen for investigation. The experimental procedure is tested for model substances with well known amounts of bound water, e.g. Na-tartratedihydrate and molecular sieve. The investigated examples show a different water sorption capacity from 4 to 60% related to the dry substance. Dehydration kinetics further demonstrate the existence of at least four types of sorbed water with distinct fractionation factors. They are characterized by the specific-activity ratio of sorbed water to free water, which ranges from 0.97 to 1.46.

Cervicovaginal synthesis of IgG antibodies to the immunodominant 175–199 domain of the surface glycoprotein gp46 of human T‐cell leukemia virus type I

Paired sera, saliva and cervicovaginal secretions from 17 HTLV-I-infected women (19–75 yr) were tested for total IgA and IgG, for IgA and IgG to the immunodominant region gp46/175-Pro-199, for serum IgG to the neutralizing domains gp46/190-Pro-199 and gp46/190-Ser-199, or for tax-rex proviral HTLV-DNA. Serum antibodies to gp46/175-Pro-199 were detected more frequently in the IgG (13/17) than in the IgA (5/17) isotypes. The majority (8/12) of anti-gp46/175-Pro-199-positive sera reacted also to gp46/190-Pro-199 or to gp46/190-Ser-199, demonstrating their neutralizing properties. In saliva, antibodies to gp46/175-Pro-199 were not generally detected. In cervicovaginal secretions, IgG to gp46/175-Pro-199, but not IgA, were detected in 6/15 (40%) patients. The mean specific activity of IgG to gp46/175-Pro-199 showed a trend to be higher in cervicovaginal secretions (218 ± 109) than in sera (14 ± 4). Furthermore, in all patients with cervicovaginal IgG to gp46/175-Pro-199, the cervicovaginal/serum ratio (19 ± 6) of anti-gp46 IgG specific activities were markedly above 1. HTLV-DNA was detected in 4/17 salivas, and in 3/15 cervicovaginal secretions, all from patients demonstrating cervico-vaginal synthesis of IgG to gp46/175-Pro-199. In conclusion, IgG to gp46/175-Pro-199 in cervico-vaginal secretions, when present, appear to be produced primarily locally because of local HTLV-I excretion. Since anti-gp46/175-Pro-199 antibodies usually support reactivities to neutralizing domains, their presence could be relevant for limiting HTLV-I transmission via cervico-vaginal secretions. © 1996 Wiley-Liss, Inc.

Cervicovaginal synthesis of IgG antibodies to the immunodominant 175-199 domain of the surface glycoprotein gp46 of human T-cell leukemia virus type I.

Paired sera, saliva and cervicovaginal secretions from 17 HTLV-I-infected women (19-75 yr) were tested for total IgA and IgG, for IgA and IgG to the immunodominant region gp46/175-Pro-199, for serum IgG to the neutralizing domains gp46/ 190-Pro-199 and gp46/190-Ser-199, or for tax-rex proviral HTLV-DNA. Serum antibodies to gp46/ 175-Pro-199 were detected more frequently in the IgG (13/17) than in the IgA (5/17) isotypes. The majority (8/12) of anti-gp46/175-Pro-199-positive sera reacted also to gp46/190-Pro-199 or to gp46/ 190-Ser-199, demonstrating their neutralizing properties. In saliva, antibodies to gp46/175-Pro-199 were not generally detected. In cervicovaginal secretions, IgG to gp46/175-Pro-199, but not IgA, were detected in 6/15 (40%) patients. The mean specific activity of IgG to gp46/175-Pro-199 showed a trend to be higher in cervicovaginal secretions (218 +/- 109) than in sera (14 +/- 4). Furthermore, in all patients with cervicovaginal IgG to gp46/175-Pro-199, the cervicogaginal/serum ratio (19 +/- 6) of anti-gp46 IgG specific activities were markedly above 1. HTLV-DNA was detected in 4/17 salivas, and in 3/15 cervicovaginal secretions, all from patients demonstrating cervicovaginal synthesis of IgG to gp46/175-Pro-199. In conclusion, IgG to gp46/175-Pro-199 in cervicovaginal secretions, when present, appear to be produced primarily locally because of local HTLV-I excretion. Since anti-gp46/175-Pro-199 antibodies usually support reactivities to neutralizing domains, their presence could be relevant for limiting HTLV-I transmission via cervicovaginal secretions.

Expression and Characterization of Human Pancreatic Preprocarboxypeptidase A1 and Preprocarboxypeptidase A2

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA inSaccharomyces cerevisiae,the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band withMr34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted inkcat/Kmvalues of 57,000 and 19,000M−1s−1for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D,L-phenyllactate, we found unique esterase/peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate–α-phenylalanine (MTX-Phe) and methotrexate–α-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. Thekcat/Kmvalues for MTX-Phe were 440,000 and 90,000M−1s−1for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000M−1s−1for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60°C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.

Effects of aging on regional rates of cerebral protein synthesis in the Sprague-Dawley rat: examination of the influence of recycling of amino acids derived from protein degradation into the precursor pool

The quantitative autoradiographic l-[1- 14C]leucine method for determination of regional rates of cerebral protein synthesis (1CPS leu) requires knowledge of the degree of recycling of leucine derived from protein degradation into the precursor pool for protein synthesis. The influence of recycling can be evaluated by measuring λ, the steady state ratio of the leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. To define the changes in 1CPS leu during the process of normal aging in the rat we have evaluated λ in middle-aged (14 months) and aged (24 months) rats and compared its values with those obtained previously in young adult rats (two months of age). The results show that the value of λ is the same in all three age groups, and that there is no change with aging in the fraction of leucine in the precursor pool derived from protein degradation. Our previously reported regional rates of protein synthesis in young adult and aged rats were based on the assumption that there was no recycling of leucine derived from protein degradation into the precursor pool for protein synthesis [Ingvar M. C., Maeder P., Sokoloff L. and Smith C. B. (1985) Brain 108, 155–170]. These values have been recalculated in the present study in order to take into account the appropriate correction for recycling. The recalculated rates are higher than those reported previously, but the effects of aging in the brain as a whole and in some specific brain regions are confirmed. Decreased 1CPS leu was observed by middle-age, and in this cross-sectional study did not appear to decrease further. Of the 39 brain regions examined decreases were found throughout the brain with some proclivity for the brain stem. In comparison with young adults the weighted average rate of protein synthesis in the brain as a whole was found to be decreased by 16 and 11 % in the middle-aged and aged groups, respectively.

Influence of valine flooding on channeling of valine into tissue pools and on protein synthesis.

Rates of valine incorporation into protein were measured under control and valine-"flooding" conditions and included correction for the degree of recycling of unlabeled valine derived from the steady-state breakdown of tissue protein into the precursor pool (tRNA bound). The correction factor lambda, which is the ratio of the steady-state specific activity of valine in the tissue tRNA-bound pool to that in the arterial plasma, was determined for each of the tissues. In controls, values of lambda ranged from 0.31 in adrenals to 0.54 in heart; in flooded animals, values were higher, but only in liver was the value of lambda close to 1.0. In control and flooded rats, rates of protein synthesis were highest in liver and adrenals and lowest in skeletal muscle, with intermediate values in brain and heart. Flooding resulted in increased rates of protein synthesis in liver and decreased rates in adrenals. Rates of protein synthesis in brain, heart, and skeletal muscle were not statistically significantly affected by flooding.

Enantioselectivity suggests a cytosolic origin for a commercial pig liver esterase preparation

A widely utilized pig liver esterase preparation has been found to be derived essentially exclusively from the cytosolic fraction of pig livers. Esterases in cytosol and microsomes prepared from a fresh pig liver hydrolyzed the S- and R-enantiomers of racemic oxazepam 3-acetate (rac-OXA) with specific activity ratios of approximately 2.3:1 and 1:62, respectively. Product formations were analyzed by chiral stationary phase high-performance liquid chromatography. The commercial pig liver esterase preparation showed greater activity toward S-OXA than did the esterases in the cytosolic fraction prepared from fresh pig liver. The results established that (i) esterases contained in microsomes and cytosol of pig liver have opposite enantioselectivity in the hydrolysis of rac-OXA and (ii) the commercial pig liver esterase preparation has a cytosolic origin.

Lactate release and uptake in hepatoma 7288CTC perfused in situ with l-[(U)- 14C] lactate or d-[(U)- 14C] glucose

Arteriovenous differences (AVD) for glucose and lactic acid measured across tissue-isolated rat tumors in vivo have shown that individual tumors with similar rates of glucose consumption may either release or utilize lactic acid. The experiments described here investigated the relationships among arterial blood lactate concentrations and tumor lactate and glucose balances. AVDs for lactate, pyruvate, glucose, 14CO 2, Po 2, Pco 2, pH, and lactate specific activities were measured across 17 tissue-isolated 7288CTC hepatomas perfused in situ with arterial blood containing 2.5 to 14.4 mmol/L lactate and either l-[(U)- 14C]lactic acid or d-[(U)- 14C]glucose. Measurements were made over a range of blood flow rates from 60% to 200% of the mean in vivo rate, 0.11 mL/min. Data collected during steady states were compared by regression analysis. Tumor lactate balance and the arterial blood lactate concentration were directly related ( r = .895, n = 22, P < .01). Net negative and positive balances occurred below and above approximately 6.5 mmol/L arterial blood lactate, respectively. The mean intratumor lactate concentration for all tumors was 6.9 ± 1.0 mmol/L (mean ± SD, n = 13). Rates of 14C-lactate oxidation to 14CO 2 ( r = .716, n = 18, P < .01) and tumor venous / arterial blood 14C-lactate specific activity ratios ( r = .845, n = 19, P < .01) were low during lactate release and were increased during lactate uptake. Total arterial blood lactate removal estimated from chemical and isotopic analyses was 23.1% ± 11% and 43.0% ± 16% ( P < .05), respectively, for six lactate-utilizing tumors. Perfusions performed with 14C-glucose showed that approximately 50% of the glucose consumed during net negative lactate balance was released as 14C-lactate to the tumor venous blood, whereas only 5% was released as 14C-lactate during net positive lactate balance. The data support the following conclusions: Arterial blood lactate controls net lactate balance in solid tumors; high concentrations increase uptake. Lactate uptake inhibits lactate formation from glucose without changing the glucose balance. Lactate is released during net lactate uptake. Since lactate uptake may exceed glucose uptake, arterial blood lactate can be a substrate for tumor energy metabolism and growth.

Radioassay of Bifunctional 5-Aminoimidazole-4-carboxamide Ribotide Transformylase-IMP Cyclohydrolase by Thin-Layer Chromatography

A radioassay has been developed for the bifunctional enzyme, AICAR transformylase-IMP cyclohydrolase, which catalyzes reactions 9 and 10 of the de novo pathway for biosynthesis of purine nucleotides (AICAR → FAICAR → IMP). 3H-labeled AICAR or FAICAR is converted enzymically to product(s) which are separated by one-dimensional thin-layer chromatography prior to quantification by scintillation counting. Using this sensitive radioassay, a dissociation constant of IMP cyclohydrolase for FAICAR of 0.87 μM has been determined and AICAR, FAICAR, and IMP can be quantified in assay mixtures for AICAR transformylase-IMP cyclohydrolase. The ratio of specific enzymic activities for AICAR transformylase:IMP cyclohydrolase is 1:44.

Myriophyllum spicatum Pumps Essential and Nonessential Trace Elements from Sediments to Epiphytes

To test the hypothesis that elements can be transferred from freshwater sediments to macrophytes and to epiphytes, we grew Myriophyllum spicatum in natural sediments containing 60Co and 54Mn (essential) and 137Cs, 152Eu, 154Eu, and 155Eu (nonessential). We found no differences between shoot or epiphyte to sediment bioaccumulation factors (BAFs) and only found differences between root and shoot to sediment BAFs for 60Co and 54Mn, probably because these elements adsorb onto root surfaces. We found no differences in BAFs for any elements between shoots and epiphytes, suggesting no differences in the transfer of essential and nonessential elements. There were, however, interelement differences for the roots, again probably due to surface adsorption related to redox sensitivity. Using isotope specific activity ratios, we calculated that over 75% of the Co and 100% of the Mn in M. spicatum was obtained from the sediments and that all Co and Mn in the epiphytes was sediment derived through the host. Although the net flux of trace elements from the sediments to the water via macrophytes has been shown to be small in most situations, buried pulses of nonessential elements may be reintroduced to the littoral food web by epiphytes being grazed by invertebrates and fish.

Evidence for brain docosahexaenoate recycling in the free-moving adult rat: implications for measurement of phospholipid synthesis

The specific activity (SA) of unesterified docosahexaenoic acid (22:6 n-3) in the brain and arterial plasma was measured after constant intravenous infusion of [ 3H] 22:6 n-3 in the free-moving rat. Within 40–105 min, an apparent steady state of labeled unesterified 22:6 n-3 in plasma and in brain was reached. However, the values of the brain to plasma 22:6 n-3 SA ratios ranged from 0.03 to 0.05, indicating that an isotopic equilibrium between brain and plasma was not attained. This suggests that a considerable endogenous source of unesterified 22:6 n-3 (95–97%) (likely derived from lipid metabolism) dilutes the SA of the tracer coming from plasma. Using the SA of 22:6 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis.

Evidence that both Ca 2+-ATPase and (Ca 2+ + Mg 2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

1. 1. Evidence was obtained that activities of both low-affinity Ca 2+-ATPase and high-affinity (Ca 2+ + Mg 2+)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme. 2. 2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant. 3. 3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca 2+-ATPase activity comigrated with that of (Ca 2+ + Mg 2+)-ATPase. 4. 4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated. 5. 5. These findings suggest that Ca 2+-ATPase and (Ca 2+ + Mg 2+)-ATPase are a single enzyme.

Skeletal muscle glycogenolysis is more sensitive to insulin than is glucose transport/phosphorylation. Relation to the insulin-mediated inhibition of hepatic glucose production.

The effects of minimal increments in plasma insulin concentrations on hepatic glucose production and glucose uptake, skeletal muscle net glycogen synthesis and glycogenolysis, glycogen synthase and phosphorylase activity, glucose-6-phosphate and uridinediphosphoglucose (UDPG) concentrations were examined in 24-h and in 6-h fasted conscious rats. Insulin was infused for 120 min at rates of 1.5, 3, 6, 12, 24, and 108 pmol/kg per min in 24-h fasted rats and at rates of 3, 6, 9, 12, 36, and 108 pmol/kg per min in 6-h fasted rats while endogenous insulin release was inhibited by SRIF infusion and plasma glucose was maintained at the basal level. All rats received an infusion of [3-3H]glucose. The portion of the muscle glucose-6-phosphate (G6P) pool derived from net glycogenolysis was estimated from the ratio of specific activities of muscle UDPG and plasma glucose. Minimal increments in the circulating insulin levels, which did not stimulate glucose uptake, caused: (a) the increase in skeletal muscle glycogen synthase activity and the decrease in the rate of muscle glycogenolysis and in the G6P concentration; (b) the inhibition of hepatic glucose production. Net muscle glycogen synthesis was not stimulated despite submaximal activation of glycogen synthase, and its onset correlated with the rise in muscle G6P levels. Thus, insulin's inhibition of muscle glycogenolysis is the most sensitive insulin action on skeletal muscle and its dose-response characteristics resemble those for the inhibition of hepatic glucose production. These findings indicate that skeletal muscle glycogen synthase may play a major role in carbohydrate homeostasis even under postabsorptive (basal insulin) conditions and support the notion that insulin may exert some of its effects on the liver through an indirect or peripheral mechanism.

14C-labeled propionate metabolism in vivo and estimates of hepatic gluconeogenesis relative to Krebs cycle flux.

Purposes of this study were 1) to estimate in humans, using 14C-labeled propionate, the rate of hepatic gluconeogenesis relative to the rate of Krebs cycle flux; 2) to compare those rates with estimates previously made using [3-14C]lactate and [2-14C]acetate; 3) to determine if the amount of ATP required for that rate of gluconeogenesis could be generated in liver, calculated from that rate of Krebs cycle flux and splanchnic balance measurements, previously made, and 4) to test whether hepatic succinyl-CoA is channeled during its metabolism through the Krebs cycle. [2-14C]propionate, [3-14C]-propionate, and [2,3-14C]succinate were given along with phenyl acetate to normal subjects, fasted 60 h. Distributions of 14C were determined in the carbons of blood glucose and of glutamate from excreted phenylacetylglutamine. Corrections to the distributions for 14CO2 fixation were made from the specific activities of urinary urea and the specific activities in glucose, glutamate, and urea previously found on administering [14C]-bicarbonate. Uncertainties in the corrections and in the contributions of pyruvate and Cori cyclings limit the quantitations. The rate of gluconeogenesis appears to be two or more times the rate of Krebs cycle flux and pyruvate's decarboxylation to acetyl-CoA, metabolized in the cycle, less than one-twenty-fifth the rate of its decarboxylation. Such estimates were previously made using [3-14C]lactate. The findings support the use of phenyl acetate to sample hepatic alpha-ketoglutarate. Ratios of specific activities of glucose to glutamate and glucose to urinary urea and expired CO2 indicate succinate's extensive metabolism when presented in trace amounts to liver. Utilizations of the labeled compounds by liver relative to other tissues were in the order succinate = lactate > propionate > acetate. ATP required for gluconeogenesis and urea formation was approximately 40% of the amount of ATP generated in liver. There was no channeling of succinyl-CoA in the Krebs cycle in the hepatic mitochondria.

NADH-ascorbate free radical and -ferricyanide reductase activities represent different levels of plasma membrane electron transport

Plasma membranes isolated from rat liver by two-phase partition exhibited dehydrogenase activities for ascorbate free radical (AFR) and ferricyanide reduction in a ratio of specific activities of 1:40. NADH-AFR reductase could not be solubilized by detergents from plasma membrane fractions. NADH-AFR reductase was inhibited in both clathrin-depleted membrane and membranes incubated with anti-clathrin antiserum. This activity was reconstituted in plasma membranes in proportion to the amount of clathrin-enriched supernatant added. NADH ferricyanide reductase was unaffected by both clathrin-depletion and antibody incubation and was fully solubilized by detergents. Also, wheat germ agglutinin only inhibited NADH-AFR reductase. The findings suggest that NADH-AFR reductase and NADH-ferricyanide reductase activities of plasma membrane represent different levels of the electron transport chain. The inability of the NADH-AFR reductase to survive detergent solubilization might indicate the involvement of more than one protein in the electron transport from NADH to the AFR but not to ferricyanide.

Limitations in the use of [2-14C]acetate for measuring gluconeogenesis in vivo.

This study was undertaken to test two assumptions critical for use of [2-14C]acetate to measure gluconeogenesis in vivo. For the assumption that incorporation into glucose of products of [14C]acetate metabolism does not affect the distribution of label within the glucose molecule, we infused [2-14C]acetate in 17 healthy subjects and [3-14C]lactate in 10 healthy subjects and compared the ratio of the resultant specific activities of plasma glucose carbons 1, 2, 5, 6, and 3, 4 obtained with each tracer. The ratio obtained with [2-14C]acetate (2.99 +/- 0.07) was significantly different from the ratio obtained with [3-14C]lactate, (3.82 +/- 0.2, P < 0.01). Because the model predicts that these ratios should be identical, these results indicate that either the model is incorrect or that metabolism of [14C]acetate to other compounds affects the distribution of the label within the glucose molecule. To test the assumption that plasma 3-OH-butyrate specific activity approximates the specific activity of hepatic intramitochondrial acetyl CoA, we compared the ratio of specific activities of plasma glucose and 3-OH-butyrate obtained in 7 healthy subjects infused with [2-14C]acetate and [2-14C]octanoate. The ratio obtained with [2-14C]acetate (0.18 +/- 0.03) was significantly different from that obtained with [2-14C]octanoate, (0.10 +/- 0.02), P < 0.001. These results suggest compartmentalization of acetyl CoA within liver mitochondria and indicate that plasma 3-OH-butyrate specific activity may not necessarily approximate intramitochondrial acetyl CoA specific activity during [2-14C]acetate infusion. We conclude that assumptions critical for use of [2-14C]acetate to measure gluconeogenesis in vivo are not valid.

Investigation of the mechanism of non-turnover-dependent inactivation of purified human 5-lipoxygenase. Inactivation by H2O2 and inhibition by metal ions.

Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.

Determination of regional rates of cerebral protein synthesis adjusted for regional differences in recycling of leucine derived from protein degradation into the precursor pool in conscious adult rats.

The quantitative autoradiographic L-[1-14C]leucine method for the determination of regional rates of cerebral protein synthesis in vivo takes into account recycling of unlabeled leucine derived from protein degradation into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the apparent steady-state leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. In the whole brain of the conscious rat this ratio (lambda WB) equals 0.58. The equivalent ratio for leucine in the acid-soluble pool in whole brain (psi WB) is 0.49. A first-degree polynomial equation for lambda WB as a function of psi WB was fitted from paired determinations. To determine the degree of recycling in local regions of the brain, we have measured in individual brain regions (i) psi i and calculated lambda i assuming that the fitted equation also applies to these localized regions. Our results indicate that the degree of recycling into the precursor pool does vary regionally; lambda i in the individual regions varies from 0.62 in the hypoglossal nucleus to 0.50 in the globus pallidus. Local rates of protein synthesis were then determined by the autoradiographic technique with regional corrections for recycling of unlabeled leucine. Rates of leucine incorporation into protein averaged 6.1 nmol/g of tissue/min in the brain as a whole, with the rates in gray matter about twice those in white matter.

In vivo effect of l-leucine administration on protein synthesis in mice

To clarify the in vivo effect of leucine on protein synthesis within a short period, the fractional formation rate of peptidyl puromycin (FFR) was measured in the liver and skeletal muscle of mice. FFR was calculated as the ratio of specific activity of peptide-bound puromycin at 10 minutes after injection of [ 3H]puromycin to the area under the specific activity-time curve of free puromycin. The value of FFR corresponds to the amount of tracer puromycin bound in unit time, expressed as a fraction of the total amount of puromycin potentially bound to the aminoacyl-site on the functional ribosome pool. In 12-hour fasted, streptozotocin-induced diabetic mice, l-leucine injection (360 μmol/100 g body weight) increased the FFR in the liver but not in muscle, while the injection of insulin (0.5 unit/100 g body weight) raised the rate in muscle but not in the liver. The leucine-induced rise of FFR in the liver was blocked by a cyclooxygenase inhibitor, indomethacin, although this inhibitor did not block the insulin-stimulated rise of FFR in muscle. No significant increase in FFR was detected in the liver or muscle of non-diabetic mice by leucine injection. l-leucine injection caused a slight decrease of ribosome aggregation in the liver but not in muscle, while insulin injection led to ribosome aggregation in muscle but not in the liver of diabetic mice. The enhancement of FFR in the liver of diabetic animals by leucine injection suggests that leucine may stimulate protein synthesis in part by increasing the rate of elongation.

Regional cerebral L-[14C-methyl]methionine incorporation into proteins: evidence for methionine recycling in the rat brain.

The specific activity (SA) of free methionine was measured in plasma and in different regions of the rat brain at 15, 30, or 60 min after intravenous infusion of L-[14C-methyl]methionine. Within these time periods, an apparent steady state of labeled free methionine in plasma and in brain was reached. However, the brain-to-plasma free methionine SA ratio was found to be approximately 0.5, showing that an isotopic equilibrium between brain and plasma was not attained. This suggests the presence of an endogenous source of brain free methionine (likely originating from protein breakdown), in addition to the plasma source. The contribution of this endogenous source to the content of free methionine varies significantly among the different brain regions. Our results indicate that the regional rates of protein synthesis measured with L-[11C-methyl]methionine using positron emission tomography would be underestimated, since the local fraction of brain methionine derived from protein degradation would not be considered.