Protein turnover and growth in the whole body, liver and kidney of the rat from the foetus to senility.

Changes in the growth and protein turnover (measured in vivo) of the rat liver, kidney and whole body were studied between 16 days of life in utero and 105 weeks post partum. Tissue and whole-body growth were related to changes in both cellular hyperplasia (i.e. changes in DNA) and hypertrophy (protein/DNA values) and to the protein composition within the enlarging tissue mass. The suitability of using a single large dose of phenylalanine for measuring the rates of protein synthesis during both pre- and post-natal life was established. The declining growth rates in the whole animal and the two visceral tissues were then explained by developmental changes in the fractional rates of protein synthesis and breakdown, turnover rates being age-for-age higher in the liver than in the kidney, which in turn were higher than those measured in the whole animal. The declining fractional rates of synthesis in both tissues and the whole body with increasing age were related to changes in the tissues' ribosomal capacity and activity. The fall in the hepatic rate between 18 and 20 days of foetal life (from 134 to 98% per day) corresponded to a decrease in both the ribosomal capacity and the rate of synthesis per ribosome. No significant changes in any of these parameters were, however, found in the liver between weaning (3 weeks) and senility (105 weeks). In contrast, the fractional synthetic (and degradative) rates progressively declined in the kidney (from 95 to 24% per day) and whole body (from 70 to 11% per day) throughout both pre- and post-natal life, mainly as a consequence of a progressive decline in the ribosomal capacity, but with some fall in the ribosomal activity also during foetal life. The age-related contributions of these visceral tissues to the total amount of protein synthesized per day by the whole animal were determined. The renal contribution remained fairly constant at 1.6-2.9%, whereas the hepatic contribution declined from 56 to 11%, with increasing age. Approximate-steady-state conditions were reached at, and between, 44 and 105 weeks post partum, the half-life values of mixed whole-body, kidney and liver proteins being 6.4, 3.0 and 1.5 days, respectively, at 105 weeks.     

Regulation of actin polymerizaton in rat islets of Langerhans.

The rate of protein synthesis was assessed in liver, stomach, small and large intestine and in the whole body of rats by injection of 100 mumol of [14C]leucine/100 g body wt. In each of the tissues turnover was very rapid, so that taken together they accounted for 43% of the protein synthesized by the whole animal.     

Influence of dietary non-essential amino acid profile on growth performance and amino acid metabolism of Nile tilapia, Oreochromis niloticus (L.)

The quality of dietary protein is an important factor influencing the growth performance of fish. To evaluate the quality of protein, the variables commonly studied are the composition of the essential amino acids, the digestibility and the protein use efficiency. The goal of the present experiment was to test the effect of the dietary non-essential amino acid composition on the growth of Nile tilapia (Oreochromis niloticus). The fish were fed three purified diets differing only in their non-essential amino acid composition. The influence of the experimental diets on the growth performance, on the activity of enzymes involved in the amino acid metabolism, aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), and on whole body delta(15)N values was investigated. Body mass, lipid, protein and energy gain differed significantly between the feeding groups. The activity of ASAT in the whole liver was significantly higher in fish with a positive protein balance compared to fish which lost protein. Whole body delta(15)N values of fish were negatively correlated with their body mass gain. Despite the poor utilisation of synthetic amino acids, the experiment indicates the importance of the dietary non-essential amino acid composition for the growth performance of fish. The study reveals the possibility to trace the utilisation of synthetic amino acids by determining the isotopic composition of dietary amino acids and tissues or whole bodies of animals.     

Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation

Summary Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions — heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material — a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin, stimulation bothin vivo andin vitro were unsuccessful.     

Variations in Both TG1 and TG2 Isozyme-specific In Situ Activities and Protein Expressions during Mouse Embryonic Development

Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. Using fluorescent-labeled highly reactive substrate peptides, we recently developed a system to visualize isozyme-specific in situ enzymatic activity. In the present study, we investigated the in situ activities of TG1 (skin-type) and TG2 (tissue-type) using whole mouse sections of various embryonic developmental stages and neonates. In each case, we also successfully used immunostaining of identical whole mouse sections for protein expression after detection of enzymatic activities. In general, the enzymatic activity was correlated with TG protein expression. However, in some tissues, TG protein expression patterns, which were inconsistent with the enzymatic activities, suggested that inactive TGs were produced possibly by self cross-linking or other modifications. Our method allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues.     

The regulation of protein synthesis in animal cells by serum factors.

We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.     

Evidence for the existence of multiple forms of choline (ethanolamine) kinase in rat tissues

In order to characterize the form of choline kinase in rat tissues, both electrophoretic and gel chromatographic patterns of choline kinase activity were compared in the liver, kidney, lung, whole intestine and carbon tetrachloride-induced liver cytosols. Kinetic parameters of the reaction were also compared for the main forms of choline kinase protein from these tissues. The overall results suggested strongly that choline kinase does not exist in one particular active form but exists in multiple forms in rat tissues. In the study present here, the electrophoretic patterns of both choline kinase and ethanolamine kinase activities were compared in rat liver, kidney, lung and intestinal cytosols. The results strongly supported the view that both kinase activities are represented on the same enzyme protein(s) in each of the rat tissues examined.     

Pre- and post-natal growth and protein turnover in smooth muscle, heart and slow- and fast-twitch skeletal muscles of the rat.

The growth of one smooth and three individual striated muscles was studied from birth to old age (105 weeks), and where possible during the later stages of foetal life also. Developmental changes in protein turnover (measured in vivo) were related to the changing patterns of growth within each muscle, and the body as a whole. Developmental growth (i.e. protein accumulation) in all muscles involved an increasing proportion of protein per unit wet weight, as well as cellular hypertrophy. The contribution of the heart towards whole-body protein and nucleic acid contents progressively decreased from 18 days of gestation to senility. In contrast, post-natal changes in both slow-twitch (soleus) and fast-twitch (tibialis anterior) skeletal muscles remained reasonably constant with respect to whole-body values. Such age-related growth in all four muscle types was accompanied by a progressive decline in both the fractional rates of protein synthesis and breakdown, the changes in synthesis being more pronounced. Age for age, the fractional rates of synthesis were highest in the oesophageal smooth muscle, similar in both cardiac and the slow-twitch muscles, and lowest in the fast-twitch tibialis muscle. Despite these differences, the developmental fall in synthetic rates was remarkably similar in all four muscles, e.g. the rates at 105 weeks were 30-35% of their values at weaning. Such developmental changes in synthesis were largely related to diminishing ribosomal capacities within each muscle. When measured under near-steady-state conditions (i.e. 105 weeks of age), the half-lives of mixed muscle proteins were 5.1, 10.4, 12.1 and 18.3 days for the smooth, cardiac, soleus and tibialis muscles respectively. Old-age atrophy was evident in the senile animals, this being more marked in each of the four muscle types than in the animal as a whole. In each muscle of the senile rats the protein content and composition per unit wet weight, and both the fractional and total rates of synthesis, were significantly lower than in the muscles of younger, mature, animals (i.e. 44 weeks). In the soleus the decreased synthesis rate appeared to be related to a further fall in the ribosomal capacity. In contrast, the changes in synthesis in the three remaining muscles correlated with significant decreases in the synthetic rate per ribosome.(ABSTRACT TRUNCATED AT 400 WORDS)     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

In vivo protein synthesis in different tissues and the whole body of rainbow trout (Salmo gairdnerii R.). Influence of environmental temperature

The fractional protein synthesis rate (FSR) of tissue (liver, digestive tract, muscle and whole fish) proteins was measured in rainbow trout acclimated to 9 and 18 degrees C after a pulse injection of [U-14C] L-leucine. In each of the tissues two FSRs were calculated based on a different estimate of the specific radioactivity of leucine in the precursor compartment for protein synthesis. Whole fish protein synthesis (WFPS) was estimated to be 7 and 7.6 g protein per kg body weight and per day respectively at 10 and 18 degrees C. Muscle and digestive tract contributed the most (more than 30%) to WFPS. The rate of protein turnover in whole fish was very low, as in the muscle, when compared to liver and digestive tract.     

Studies on a peptidase acting on a synthetic collagenase substrate Developmental pattern in rat granuloma tissue and distribution of enzyme in the tissues of various animals

The developmental pattern in experimental rat granuloma tissue and the distribution in the tissues of a few animals (monkey, rabbit, guinea pig anrat) of a peptidase acting on a synthetic collagenase substrate, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) has been studied. Maximum enzyme activity was found in 4-month-old rats and on the fourth day of implantation of the cotton wick. Pz-peptidase appears to have a ubiquitous distribution in animal tissues; the highest enzyme activity was generally found in liver, intestine and kidney of the animals. The total activity in other organs (spleen, heart, lungs and brain) was much less compared to that of liver, intestine or kidney.     

Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.     

Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2 signaling

Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.     

Human protein metabolism: its measurement and regulation

The body's protein mass not only provides architectural support for cells but also serves vital roles in maintaining their function and survival. The whole body protein pool, as well as that of individual tissues, is determined by the balance between the processes of protein synthesis and degradation. These in turn are regulated by interactions among hormonal, nutritional, neural, inflammatory, and other influences. Prolonged changes in either the synthetic or degradative processes (or both) that cause protein wasting increase morbidity and mortality. The application of tracer kinetic methods, combined with measurements of the activity of components of the cellular signaling pathways involved in protein synthesis and degradation, affords new insights into the regulation of both protein synthesis and breakdown in vivo. These insights, including those from studies of insulin, insulin-like growth factor I, growth hormone, and amino acid-mediated regulation of muscle and whole body protein turnover, provide opportunities to develop and test therapeutic approaches with promise to minimize or prevent these adverse health consequences.     

RIBOSOMAL ACTIVITY IN PRENATAL MOUSE BRAIN

Abstract— Regulation of protein synthesis is important for the proper growth and development of the brain. Our previous work on the regulation of protein synthetic activity in fetal mouse brain cell suspensions showed that the rate of protein synthesis decreased during the prenatal period. In the present study, ribosomal activity of cell-free homogenates and purified ribosomes obtained from fetal neural tissue was measured. The post-mitochondrial supernatant (PMS) fraction actively incorporated amino acids into polypeptides using either endogenous mRNA or polyuridylic acid as template. The protein synthetic activity was dependent upon the age of the fetus. Ribosomes purified from this fraction were also active in protein synthesis. Incorporation of phenylalanine was linear for 20 min, and dependent upon the concentration of ribosomes and the pH 5 enzyme fraction. The age dependent decrease in protein synthetic activity observed with the post-mitochondrial supernatant fractions was not found when these purified ribosomes were employed. Ribosomes obtained from fetal, newborn or adult neural tissue were compared and found equally active in their protein synthetic capacity.     

Protein synthesis in the whole body,liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis

Jones W. O. and Symons L. E. A. 1982. Protein synthesis in the whole body, liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis. International Journal for Parasitology12: 295–301. Tyrosine flux and the synthesis of protein in the whole body, liver, skeletal muscle and kidney cortex and of albumin in lambs infected with Trichostrongylus colubriformis and uninfected lambs fed ad libitum or pair-fed with the infected group, were measured by constant infusion of ¹⁴C-l-tyrosine. Live weight gain was lower in the infected than in pairfed lambs, but rates of whole body protein synthesis were similar in both groups. On the other hand, compared with control lambs, there was a faster rate of protein synthesis per unit of protein consumed in infected but not in pair-fed lambs. Rates of protein synthesis per unit of body weight in infected were higher than in pair-fed lambs, but similar to the rate in control lambs. The fractional synthetic rates (FSR) of albumin and liver proteins and the amount of liver protein synthesized per day were increased by infection. The FSR and amount of protein synthesized per day were depressed in skeletal muscle and kidney cortex. Anorexia did not explain any of these changes. Infection caused a loss of protein from each of these tissues, but this loss was due to anorexia in only the liver. There was generally good correlation between concentration of RNA per g fresh weight or per mg nitrogen and the FSR of protein. However, although the $\text{RNA}\text{DNA}$ ratio correlated well with synthesis in skeletal muscle, it was poorly correlated for liver proteins. The relationship between the rate of growth and protein synthesis in infected lambs is discussed.     

Protein synthesis in the organs and tissues of frogs with local exposure to low temperatures]

Intensity of synthesis of total proteins and spectrum of synthesized proteins has been studied in various organs and tissues of sharp-faced frogs during local profound cooling of one of its limbs. It was shown that the effect of low temperatures on a certain part of a body of a poikilothermic animal has induced both a response of the whole organism and a much more acute response of the cooled tissues. The organism responded by a fall of the whole body temperature and a decrease in the activity of synthesis of total proteins of the viscera. Local cooling of a part of the frog body has induced a more significant decrease in the protein synthesis intensity in the cooled tissues alteration in the spectrum of synthesized proteins and appearance of specific proteins.     

Effect of cholesterol feeding and fasting on sterol synthesis in seventeen tissues of the rat

Rates of sterol synthesis were measured in 17 tissues of the rat, and the responsiveness of these rates to cholesterol feeding and to fasting was determined. The liver and gastrointestinal tract together account for 90% of synthetic activity of the whole body. After the rats had been fed cholesterol or fasted, liver synthesis was markedly decreased, whereas synthetic rates in all other organs tested were essentially unaffected (this conclusion applies to synthesis of cholesterol and of five other digitonin-precipitable tissue sterols). Consequently, the highest rate of cholesterogenesis in the cholesterol-fed or fasted rat is found in the gastrointestinal tract.     

Ornithine decarboxylase activity in relation to DNA synthesis in mouse interfollicular epidermis and hair follicles.

The relationship between ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) activity and DNA synthetic activity was studied in mouse epidermis. Interfollicular epidermis and hair follicles were investigated separately. It was found that, in hair follicles, the variations of DNA replicative activity, which are reflected in the cyclic growth of hair, are paralleled by corresponding changes in ornithine decarboxylase activity. In both interfollicular epidermis and hair follicles, stimulation of DNA synthetic activity by plucking of hair induced a rapid and marked increase in ornithine decarboxylase activity. The relationship of steady-state and induced ornithine decarboxylase activity to DNA synthetic activity was compared in hair follicles and interfollicular epidermis. A correlation between the activity of this enzyme and DNA replication was found thereby in each of these tissues.