Hybrid protein synthetic systems: components required to confer poikilothermy to the mammal.

Temperature dependency of homologous and heterologous protein synthetic cell-free systems reveals characteristic homeotherm and poikilotherm behavior depending on the source of the enzyme elongation factor 1 and not on any other protein synthetic components. The results for rat and Antarctic fish, when compared with previous studies in eurythermal fish and mammals provide the explanation for the phenomenon of true hibernation in animals such as ground squirrels and woodchucks. The killing of these rare animals can now be stopped by granting agencies that support hibernation research. Comparison of temperature effects on laboratory rats and wild rats indicate a need for wild rat research directed toward eradication of these pests in cities. Suggestions are made for this purpose.

Comparative analysis of leucine transport in temperate fish liver in vivo.

The uptake of [14C]leucine in toadfish (Opsanus tau) liver in vivo at 10 degrees C has been studied by a single pulse injection technique. Transport parameters were determined on the basis of the distribution of the amino acid and of [3H]inulin, used as a marker for extracellular space, in liver free and protein-bound fractions and in venous blood draining from the liver. Saturation analysis by the Cornish-Bowden method yielded a maximal uptake of 0.26 mumole, which was similar on a concentration basis to that at 21 degrees C when circulation rate and dilution with blood are taken into account. Isoleucine and phenylalanine competed with leucine uptake at 10 degrees C as at 21 degrees C; additional competitors at 10 degrees C included histidine, methionine and valine. Fish acclimated to 10 degrees C for 2 weeks or more showed a restoration in maximal leucine uptake and disappearance of histidine inhibition. Methionine inhibition was retained. Three transport systems in this species are discussed: 20-20, operating in 20 degrees C-acclimated fish at 20 degrees C; 20-10, in 20 degrees C-acclimated fish at 10 degrees C; and 10-10, in 10 degrees C-acclimated fish at 10 degrees C. The properties of these systems are compared with the 0-0 system of Antarctic fish and with transport systems of mammalian cells. The latter are similar to our non-acclimated system, 20-10, suggesting that the mammalian cell may not be at a state of optimal temperature adaptation.

A comparative study of protein synthesis in nototheniids and icefish at Palmer Station, Antarctica.

Protein synthetic rates were measured in tissues of Notothenia corriceps, N. gibberifrons and Chaenocephalus aceratus in vivo at 2 degrees C by a method in which high doses of 14C-phenylalanine are used for stabilization of specific radioactivity. Rates in N. coriiceps, as per cent of tissue protein synthesized per day, were: liver 10.4, head kidney 3.5, testis 2.6, spleen 2.1, kidney 1.9, gill 1.6, heart 1.4, pectoral muscle 1.0, epaxial muscle 0.37, brain 0.42. With the exception of liver and head kidney (9.8 and 3.4, respectively) all rates in the icefish C. aceratus were significantly reduced compared to the nototheniids, consistent with the dependence of protein synthesis on oxidative metabolism. Icefish lack hemoglobin in the blood. The effects of two-week starvation were tissue-specific. Rates declined markedly in pectoral and epaxial muscle, were unchanged in liver, kidney, brain, heart and testis, and were increased in gill and head kidney. The results are discussed in relation to cold adaptation of Antarctic fishes and to the adaptation of metabolism required during non-feeding periods and for species which lack an oxygen-binding pigment in their blood.

Antifreeze glycopeptides of antarctic fishes.

A method is described for analysis of amino acid composition and concentration of antifreeze glycopeptides of Antarctic fish by automatic amino acid analysis of hydrolyzed samples purified by trichloroacetic acid fractionation. Preparations were 98% pure and showed characteristic alanine, threonine and proline compositions. Analyses were carried out in 17 known species of Ross Sea, Antarctica, fishes and in 11 undescribed species tentatively placed in genera, where possible, or families. Collections have been preserved for classification. The results provide evidence of species specificity in amino acid composition and plasma concentration of these substances. Closely related species showed nearly identical Pro/Ala ratios, but differing concentrations. The results do not support reclassification of Trematomus and Notothenia into a single genus or the inclusion of the genus Pleuragramma in the family Nototheniidae. Data obtained for developing ovaries of 6 species showed concentrations of these substances (per unit wet wt) about one-half those of plasma and similar, but not identical, amino acid compositions to those of plasma. The total amount in eggs was 4 times that of plasma in pre-spawning individuals. Calculations are presented to indicate the role of these substances in the protein metabolic economy of the animal for Nototheniidae and Channichthyidae.

Quantitative protein synthetic rates in various tissues of a temperate fish in vivo by the method of phenylalanine swamping.

1. A single high-dose injection of 14C- (or 3H-) phenylalanine was used for measurement of protein synthetic rates in tissues of toadfish, Opsanus tau, in vivo. 2. Average rates at 20 degrees C, as per cent of tissue protein synthesized per day, were as follows: liver, 14; head kidney, 15; gill, 7.7; spleen 5.0; heart, 2.3; testis, 1.4; epaxial and pectoral muscle, 0.20-0.23. Elevated rates in muscle (0.71%/day) were found in fed fish. 3. Temperature dependency (Q10) in the 10-20 degrees C range averaged 6-7 for tissues with high rates of protein turnover, and 3-4 in heart and muscle after two-weeks acclimation.

Characteristics of leucine transport by isolated hepatocytes of Antarctic fish at low temperatures.

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [14C]leucine at 0, 5, and 10 degrees C. The system is saturable with apparent Km about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0-10 degrees C range yielded Ea = 65 kJ/mol (Q10 = 2.7). The average first-order rate constant at 0 degrees C was 0.1 min-1, one-third the value of 0.3 min-1 estimated for clearance of [14C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0 degrees C and in temperate fish acclimated to 10 degrees C and 20 degrees C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.

L-leucine transport in liver of Antarctic fish in vivo at 0 degrees C.

Uptake of L-[14C]leucine by liver of an endemic Antarctic fish, Trematomus hansoni, was studied by a single injection technique with [3H]inulin as a reference. Rate constants for leucine influx and efflux and incorporation into liver protein were determined by analysis of isotope distribution in the free and protein-bound compartments of liver and in blood draining the liver at various times after injection. Transport rates were slower than in temperate fish at 20 degrees C, but saturation properties and ability to accumulate leucine in liver were comparable. Kinetic analysis indicated that 30% of uptake at 0 degrees C was due to active transport, similar to that in toadfish at 20 degrees C. This contrasts with the absence of this component in toadfish cooled to 10 degrees C. Average polypeptide chain assembly time was 19 min at 0 degrees C. Transport functions were maintained in fish warmed to 10 degrees C; however, protein synthesis declined at temperatures above 5 degrees C. The results indicate this system is adapted to function at extremely low temperatures in a manner qualitatively similar to organisms adapted to much higher temperatures. Transport and synthetic rates, however, were low, consistent with a normal temperature dependency (Q10 about 2.5) for biological reaction rates.

Effects of temperature on L-leucine transport in toadfish liver in vivo.

The effect of body temperature in the 4--30 degrees C range on L-leucine uptake by toadfish liver in vivo was examined by means of a single-injection pulse technique. The ratio of [14C]leucine to [3H]mannitol or [3H]inulin in blood leaving the liver was measured as a function of time after hepatic portal vein injection. Recoveries of the two isotopes in liver and [14C]leucine incorporation into protein were determined. The Q10 value for influx was 3.8, that for efflux 2.8. At all temperatures, the leucine influx was 8--10-times higher than its incorporation into protein. The directly energy-linked reactions appear to be the main site of increased temperature sensitivity at low temperatures.

An analysis of fish genomes by density gradient centrifugation.

DNA was prepared from 33 species of fishes representing 12 of the 31 orders of Teleostei and one order of Chondrichthyes. DNA samples were investigated to determine modal and mean buoyant densities in CsCl (Rho-0 and ), main-band asymmetry, intermolecular compositional heterogeneity and base composition. Modal buoyant densities ranged over 1.697-1.704 g/cm3, generally with identical or close values for DNAs from species belonging to the same order. Main bands were practically symmetrical in most cases; when present, asymmetries were on the heavy side of the band and rather modest. In most cases, compositional heterogeneities were equal to, or lower than, those of bacterial DNAs. Both the CsCl band symmetry and the low compositional heterogeneity strikingly distinguish fish DNAs from the DNAs of warm-blooded vertebrates and indicate a major discontinuity in genome organization in the vertebrates. The overall properties of the main bands of fish DNAs are very similar to those exhibited by the light components of DNAs from mammals and birds.

Protein synthesis in liver, muscle and gill of mullet (Mugil cephalus L.) in vivo.

1. Protein synthesis in mullet was measured by use of pulse injections of 14Cleucine into the hepatic portal circulation and by 4-hr continuous infusion of 14Ctyrosine into the dorsal aorta. 2. Liver protein synthesis was 37 mg/day per g wet weight of tissue. Plasma proteins accounted for about 25% of total liver protein synthesis. Replacement rate for intrahepatic proteins was 20%/day. Average polypeptide chain synthesis time was 3 min. 3. Gill protein synthesis was comparable to that in liver with a replacement rate of 23%/day. 4. Muscle protein synthetic rate was 0.54%/day, comparable to rates in rabbit and sheep when temperature difference is taken into account. 5. Constant infusion is shown to be a feasible technique for measurement of protein metabolism of fish in vivo.

Effect of temperature on protein synthesis in fish of the Galapagos and Perlas Islands.

1. The temperature dependency of protein synthesis was studied in vivo in five species of Pacific fish collected in the Galapagos and Perlas Islands: batfish (Ogcocephalus darwini), groupers (Epinephelus labriformis), catfish (Netuma platypogan), puffers (Arothron hispidus) and triggerfish (Sufflamen verres). 2. Liver protein synthesis, assayed by a rapid pulse injection technique, showed a moderate temperature dependency (Q10 = 2-3) in the 15-30 degree C range for all species except puffers (Q10 = 10-20). Synthesis was inhibited above 32 degrees C. 3. Protein synthesis in triggerfish was measured by the constant-infusion technique. Synthetic rates (% of tissue protein synthesized per day) at 25 degrees C were 20% for liver, 10% for gill, 1.8% for red muscle and 0.6% for white muscle. Q10 in the 20 degrees-30 degrees C range was 3.0 +/- 0.5 degrees C for all tissues.

Characterization of leucine transport by toadfish liver in vivo.

Kinetic analysis of L-leucine uptake by toadfish liver at 20 degrees C in vivo has been carried out after pulse injection of L-[14C]leucine into the hepatic portal vein. D-[3H]mannitol, which is taken up slowly by toadfish liver, is used as a marker for extracellular space and space accessible by simple diffusion. At normal plasma leucine concentration (0.1 mM), leucine uptake occurs rapidly (t1/2 = 0.25 min), representing a flux of 0.6 mumol/min for the liver as a whole. Analysis of the distribution of radioactive leucine among intracellular and extracellular free pools and protein-bound form at times of 30 s to 5 min after injection is consistent with operation of a concentrative or uphill transport system accounting for 40% of uptake at normal plasma concentration. Saturation of uptake occurs at increasing leucine loads; calculation of intracellular pool dilution from protein synthesis data indicates that 20-30% of liver intracellular space is occupied by incoming leucine during the first 2 min after portal injection. Maximal flux (V max) is 4.1 mumol/min per 7-g liver as a whole with Km = 0.6 mM. Competitive inhibition of leucine uptake is afforded by isoleucine and phenylalanine with lesser effects by aspartic acid, cysteine, methionine, threonine, tyrosine, and valine. No effect is observed with alanine, glycine, histidine, lysine, and proline.

Liver protein synthesis. Molecular weight distribution of pulse-labeled polypeptide chains in normal and thyroidectomized rats.

Equations are presented for determination of elongation rate in vivo for a heterogenous population of polypeptide chain molecular weights. The distribution of pulse-labeled polypeptide chains in rat liver deoxycholate-soluble protein has been obtained by sodium dodecyl sulfate-gel electrophoresis and used to compute a theoretical curve for determination of synthesis time of a 50000 mol. wt. polypeptide chain (tc50). Values of tc50 for normal and thyro-parathyroidectomized Long-Evans male rats were 1.2 and 1.75 min, respectively, representing protein synthetic rates of about 7.5 and 5.1 mg protein/g liver/h. No difference in the molecular weight profile of liver polypeptide chains on the basis of labeling or Amido-black staining was observed between the two groups. The distributions of radioactivity before and after secretion of labeled plasma protein are compared. The role of protein-synthetic rate in the changing enzyme levels associated with thyroid hormone is discussed.

Phenylalanyl-tRNA synthetases of rat liver: differential effects of thyroid hormone.

Thyroxine and analogues inhibit rat liver aminoacyl-tRNA synthetase activity for phenylalanine and tyrosine. A high yield purification of the major cytoplasmic form of phenylalanyl-tRNA synthetase (C1) and its characterization is reported. Polyribosome-bound and other sedimentable forms are found to be indistinguishable from soluble enzyme by immunoprecipitation. Mitochondrial phenylalanyl-tRNA synthetase (M) and cytoplasmic activity (C2) resistant to anti-C1 antibody have been partially purified and characterized. Tissue levels of the three forms are estimated at 22, 1.8, and 4.1 units/g of liver for C1, C2, and M, respectively [1 unit = 1 nmol of Phe-tRNA/min, 30 degrees C]. Charging capability toward rat liver and yeast tRNA, kinetic parameters, and physical properties are compared. Only enzyme C1 is hormone inhibited [K1 = 4 x 10(-6) M for triiodothyronine]. The data indicata that C2 and M are not structurally related to C1; C2 may represent an independent cytoplasmic pool of M. Implications of C1 inhibition in relation to effects on liver protein synthesis are discussed.