Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta)

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.     

Protein synthesis by attached pulmonary macrophages effect of phagocytosis

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per μg polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.     

Protein synthesis by attached pulmonary macrophages. Effect of phagocytosis

We studied the effect of phagocytosis of polystyrene latex beads on protein synthesis by pulmonary macrophages. To do this we determine the specific radioactivity of extracellular and intracellular free phenylalanine and of phenylalanine released from tRNA and used this information in calculating the rates of protein synthesis. Phagocytosis resulted in an increased rate of protein synthesis irrespective of which precursor specific radioactivity was used in the calculation. The rate of protein synthesis was increased per microgram polyribosomal RNA; but there was no increase in the amount of polyribosomal RNA in phagocytizing macrophages. The increase in the rate of protein synthesis (1.4-fold) was almost identical to the increase (1.3-fold) in the rate of ribosome transit in phagocytizing compared to nonphagocytizing macrophages. The decreased ribosome transit time during phagocytosis occurred without a fall in the average molecular weight of macrophage proteins. We conclude that phagocytosis increases the rate of protein synthesis in attached pulmonary macrophages and that this increased rate of synthesis can be accounted for almost completely by an increased rate of polypeptide chain elongation and/or termination.     

Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

Ethanol-induced inhibition of ventricular protein synthesis in vivo and the possible role of acetaldehyde

We have determined the extent to which acute ethanol administration perturbs the synthesis of ventricular contractile and non-contractile proteins in vivo. Male Wistar rats were treated with a standard dose of ethanol (75 mmol kg^?1 body weight; i.p.). Controls were treated with isovolumetric amounts of saline (0·15 mol 1^?1 NaCl). Two metabolic inhibitors of ethanol metabolism were also used namely 4-methylpyrazole (alcohol dehydrogenase inhibitor) and cyanamide (acetaldehyde dehydrogenase inhibitor) which in ethanol-dosed rats have been shown to either decrease or increase acetaldehyde formation, respectively. After 2·5 h, fractional rates of protein synthesis (i.e. the percentage of tissue protein renewed each day) were measured with a large (i.e. ‘flooding’) dose of L -[4-³H]phenylalanine (150 μmol (100 g)^?1 body weight into a lateral vein). This dose of phenylalanine effectively floods all endogenous free amino acid pools so that the specific radioactivity of the free amino acid at the site of protein synthesis (i.e. the amino acyl tRNA) is reflected by the specific radioactivity of the free amino acid in acid-soluble portions of cardiac homogenates. The results showed that ethanol alone and ethanol plus 4-methylpyrazole decreased the fractional rates of mixed, myofibrillar (contractile) and sarcoplasmic (non-contractile) protein synthesis to the same extent (by approx. 25 per cent). Profound inhibition (i.e. 80 per cent) in the fractional rates of mixed, myofibrillar and sarcoplasmic protein synthesis occurred when cyanamide was used to increase acetaldehyde formation. There was also a significant decrease in cardiac DNA content. The results suggest that acute ethanol-induced cardiac injury in the rat may be mediated by both acetaldehyde and ethanol.     

The chemical characterization of the radioactive products derived from [[3H]PheB1]insulin in the circulation of the rat.

1. Gel filtration of rat plasma taken 1 h after subcutaneous injection of [[3H]PheB1]insulin gives three peaks of radioactivity. 2. The material in these peaks was characterized by electrophoresis and chromatography. 3. We conclude that [[3H]PheB1]insulin is rapidly degraded to free tritiated phenylalanine. The phenylalanine is subsequently used for synthesis of plasma proteins de novo.     

The inhibitory action of L-dihydroxyphenylalanine (L-dopa) in the presence of a monoamine oxidase inhibitor on protein synthesis in vivo and the partial amelioration of this by methionine supplementation.

Male Wistar rats of various age groups were injected daily over a period of 3 weeks with iproniazid (10 micrograms/g body wt.) and L-dihydroxyphenylalanine (L-dopa; 0.1 mg/g body wt.). On the final day 1 h before the termination of the experiment the animals were injected with L-[14C]valine (0.1 microCi/g body wt.). The specific radioactivity of the valine in the proteins of the subcellular fractions of the tissues examined, relative to the time-integrated mean specific radioactivity of this amino acid in the acid-soluble pools of these tissues, was used to assess protein synthesis. The L-dopa/monoamine oxidase-inhibitor treatment was associated with 30--40% inhibition of protein synthesis. Supplementation of the dietary methionine intake by injection of this amino acid markedly diminished the inhibitory action of the L-dopa/monoamine oxidase-inhibitor treatment on protein synthesis in all fractions examined.     

The diurnal response of muscle and liver protein synthesis in vivo in meal-fed rats

1. The rate of protein synthesis in rat tissues was measured by constant intravenous infusion of [(14)C]tyrosine. A modification has been developed for the method of calculating the rate of protein synthesis in individual tissues from the specific radioactivity of the free and protein-bound amino acid in tissue at the end of the infusion. This technique gives greater accuracy and allows a greater choice of labelled amino acids. The specific radioactivity of free tyrosine in plasma was used to calculate the plasma tyrosine flux, an index of the rate of protein synthesis in the whole body. 2. Young male Wistar rats were allowed access to food for only 4h in every 24h. The tyrosine flux and the rate of protein synthesis in liver and muscle at different periods of time after a single feed were estimated. 3. The tyrosine flux did not alter after feeding nor even after starvation for 48h. 4. The average fractional rate of protein synthesis in muscle was 7.2%/day, i.e. the proportion of the protein mass which is replaced each day. The rate rose after eating and declined during starvation for 48h. In addition the rate of muscle protein synthesis correlated with the growth rate of the rat. 5. In liver the average fractional rate of protein synthesis was 50%/day. There was no change in the rate after eating nor after starvation for 48h. In contrast with muscle this suggests that the changes in protein mass were accompanied by changes in the rate of protein breakdown rather than synthesis.     

Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells.

A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.     

Amino acid depletion in the blood and brain tissue of hyperphenylalaninemic rats is abolished by the administration of additional lysine: a contribution to the understanding of the metabolic defects in phenylketonuria

The elevated phenylalanine concentration in the blood of untreated phenylketonuric children is known to be paralleled by decreased concentrations of other amino acids in the blood and brain tissue. Due to the low availability of other large, neutral amino acids in the brain, protein synthesis in, and the normal development of, the brain are disturbed. A similar effect is observed in suckling rats rendered hyperphenylalaninemic by the daily injection of phenylalanine plus alpha-methylphenylalanine, an in vivo inhibitor of the phenylalanine-hydroxylating pathway in the liver. In this study, the simultaneous injection of lysine is shown to prevent the depletion of amino acids from the blood and brain tissue, and the retardation of brain growth, in suckling hyperphenylalaninemic rats. It is suggested that both amino acids, phenylalanine and lysine, are important rate-limiting substrates for the rapid protein anabolism of developing tissues. In the presence of an excess of phenylalanine, other amino acids, and in relation to its requirement during the phase of hyperplastic growth in particular lysine, are less available from the circulation and limit phenylalanine-stimulated protein synthesis in developing tissues. The supplementation of lysine to developing hyperphenylalaninemic rats prevents the consequences of this effect, i.e., the depletion of amino acids in the blood, and therefore, in the brain tissue, and the retardation of brain growth.     

The mechanism of polyribosome disaggregation in brain tissue by phenylalanine.

The injection of neonatal mice with phenylalanine resulted in a rapid decrease in brain polyribosomes and a concomitant increase in monomeric ribosomes. Animals of 1-16 days of age were equally affected by phenylalanine, although the brain polyribosomes of 60-day-old mice were relatively resistant to the effects of phenylalanine. The population of free polyribosomes appeared to be more sensitive to phenylalanine treatment than bound polyribosomes, which were somewhat more resistant to disruption by high concentrations of the amino acid. The effects of phenylalanine were more pronounced with polyribosomes in the cerebral cortex than with those in the cerebellar tissue. The mechanism of polyribosome disruption was shown to be independent of hydrolysis mediated by ribonuclease. Virtually all of the monomeric ribosomes that resulted from phenylalanine treatment were shown to be inactive with regard to endogenous protein synthesis and were present in the cell cytoplasm as vacant couples. These ribosomes were readily dissociated by treatment with 0.5 M-KCl and subsequent ultracentrifugation. These results are discussed in the light of the possibility that high concentrations of phenylalanine disrupt brain protein synthesis by a molecular mechanism that is associated with initiation events.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

A simple method for measuring immunoglobulin synthesis in vivo in the spleen of chicks (Gallus domesticus)

1. Spleen immunoglobulin (IgG) synthesis was measured in vivo in chicks at 3 weeks of age by a large dose injection of labelled phenylalanine in combination with the isolation of IgG by immunological precipitation against anti-chicken IgG. 2. No appreciable amount of radioactivity was detected in serum IgG for the first 60 min of the isotope injection via the wing vein, indicating a minimum time lag necessary for the secretion of newly synthesized IgG into the circulation. 3. Synthesis rates of total spleen protein and spleen IgG were found to be 17.5 and 4.8 mg/day, respectively, suggesting that IgG synthesis would contribute to 27% of the total protein synthesis in the spleen of young chicks.     

Phenylalanine Ammonia-Lyase in Sliced Sweet Potato Roots

A marked rise in the phenylalanine ammonia-lyase activity and the polyphenol synthesis was observed in sliced roots of a sweet potato. The enzyme activity was found to be localized in the root tissue adjacent to the sliced surface. In this region, the synthesis of polyphenols was much higher compared to the inner tissues. When the specific inhibitors for the protein and nucleic acid biosynthesis such as an actinomycin D and blasticidin S were added to the tissues by vacuum infiltration technique, both the development of phenylalanine ammonia-lyase activity and the synthesis of polyphenols were severely prevented. These results suggest the important role of phenylalanine ammonia-lyase in the polyphenol synthesis and de novo synthesis of the enzyme protein molecule in the sliced tissues.     

An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid.

Rates of protein synthesis in tissues can be calculated from the specific radioactivity of free and protein-bound amino acids at the end of a constant infusion of a labelled amino acid (Garlick, Millward & James (1973) Biochem. J. 136, 935--945]. The simplifying assumptions used in these calculations have been criticized [Madsen, Everett, Sparrow & Fowkes (1977) FEBS Lett. 79, 313--316]. A more detailed analysis using a programmable desk-top calculator is described, which shows that the errors introduced by the simplifying assumptions are small, particularly when the specific radioactivity of the free amino acid rises rapidly to a constant value.     

Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis

Muscle protein synthesis in dogs measured by flooding with L-[(2)H(5)]phenylalanine (70 mg/kg) was significantly stimulated by infusion of insulin with amino acids. The stimulation of muscle protein synthesis was similar when calculated from the enrichment of phenylalanyl-tRNA (61 +/- 10%, P < 0.001), plasma phenylalanine (61 +/- 10%, P < 0.001), or tissue fluid phenylalanine (54 +/- 10%, P < 0.001). The time course for changes in enrichment of L-[(2)H(5)]phenylalanine throughout the flooding period was determined for plasma, tissue fluid, and phenylalanyl-tRNA in the basal state and during the infusion of insulin with amino acids. Enrichments of plasma free phenylalanine and phenylalanyl-tRNA were equalized between 20 and 45 min, although the enrichment of phenylalanyl-tRNA was lower at early time points. Rates of muscle protein synthesis obtained with the flooding method and calculated from plasma phenylalanine enrichment were comparable to those calculated from phenylalanyl-tRNA and also to those obtained previously with a continuous infusion of phenylalanine with phenylalanyl-tRNA as precursor. This study confirms that, with a bolus injection of labeled phenylalanine, the enrichment of aminoacyl-tRNA, the true precursor pool for protein synthesis, can be assessed from more readily sampled plasma phenylalanine.     

Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo

The turnover of prolyl hydroxylase and an immunoreactive protein that corresponds in size to the smaller subunit of the enzyme was studied in vivo after injection of [(3)H]leucine into 11-day chick embryos. The specific radioactivity and total radioactivity of the monomer-size protein were much higher than those of the enzyme tetramers in the cartilaginous bone at 3h and 12h after the radioisotope injection, indicating that the monomer-size protein represents precursors rather than degradation products of the enzyme tetramers. Between 24 and 144h after the injection the specific radioactivity and total radioactivity of the two forms of the enzyme protein showed essentially identical decay rates, the observed specific radioactivity of the monomer-size protein being about 120-130% and total radioactivity about 80% of that of the enzyme tetramers. The true half-life, when corrected for dilution caused by tissue growth and re-utilization of the [(3)H]leucine, was 37.9h for the monomer-size protein and 39.0h for the tetramers. The results obtained in the lung were less reliable owing to high blank radioactivity values in the immunoprecipitation, but even so some definite differences were found between this tissue and the cartilaginous bone. The specific radioactivity of both forms of the enzyme protein at 24h was only about 20-25% of that in the cartilaginous bone. The total radioactivity of the monomer-size protein in the lung remained about 5 times that of the enzyme tetramers, whereas it was only about 0.8 times that of the tetramers in the cartilaginous bone. As in the cartilaginous bone, the decay rates of both forms of the enzyme protein were essentially identical in the lung, with a true half-life of about 46h. The results suggest that the rate of prolyl hydroxylase synthesis is slower in the lung than in the cartilaginous bone, whereas the degradation rates are fairly similar in these two tissues. The data further suggest that, in the lung at least, a large part of the monomer-size protein became degraded without being converted into enzyme tetramers.