Seasonal pattern of glycosylation in frog liver

The circannual behaviour of glycosylation and protein synthesis in frog liver slices was studied following the incorporation of³H-galactose and¹⁴C-glucosamine into glycolipids and glycoproteins and³H-leucine into proteins. The activity of two enzymes the galactosyl-transferase and the N-acetyl-glucosaminyl-1-P-transferase was determined. The incorporations of both sugars into the soluble fraction and into the lipid extract present a maximum during the spring-summer period. The incorporation into the protein fraction displays a different pattern:¹⁴C-Glucosamine and³H-leucine incorporation increases from winter to a maximum in autumn; the incorporation of³H-Galactose has a sharp peak during spring. The pattern of glycosyltransferase activities is similar to the pattern of incorporation of the two saccharides into proteins, indicating these enzymes as important control points for glycosylation in Anurae.     

Subcellular localization of γ-glutamyl carboxypeptidase and of folates

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [³H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions.The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85–90% folate polyglutamates.Injected [³H] folic acid was initially concentrated in the microsomal fraction, as measured by ³H cpm per ng folate.Initially, injected [³H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total ³H cpm determined to be [³H] folate penta- and hexaglutamates. At 24 h, the conversion of [³H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates.Injected [³H] folic acid after 2 h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Metabolism of tritiated water in the cell nucleus and intracellular substances of the rat

Tritiated water was given to rats in single oral doses, and the cell fractions for each organ were prepared by ultracentrifugation for measurement of the concentration of tissue-bound tritium. The concentration of tissue-bound tritium reached a peak relatively soon after intubation, 1-4 days after administration. The initial concentration of tissue-bound tritium in liver and kidney was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial tissue-bound tritium concentration in the brain was high in the mitochondrial and microsomal fractions but low in the nuclear and cytosol fractions. The initial concentration of tissue-bound tritium in the testes was high in the mitochondrial, microsomal, and cytosol fractions but low in the nuclear fraction. The half-life for the long component was larger in the nuclear, mitochondrial, and microsomal fractions of the brain than in the other organs according to an interorgan comparison of each fraction. As for the testes, the values for the mitochondrial and microsomal fractions were larger than those for the other organs.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

The biosynthesis of cytochrome c. Sequence of incorporation in vivo of [14C]lysine into cytochrome c and total proteins of rat-liver subcellular fractions

1. In order to determine the initial intracellular site of synthesis of cytochrome c in the liver cell, groups of rats were injected with [(14)C]lysine and killed 7.5, 15, 30 and 60min. later. The livers were homogenized in 0.3m-sucrose and subcellular fractions obtained. The mitochondrial fraction was further subfractionated. Pure cytochrome c was isolated from extracts of each fraction, obtained first with water at pH4.0 and then with 0.15m-sodium chloride. 2. A comparison of the kinetics of incorporation of [(14)C]lysine into total protein for each particulate fraction showed the usual two different kinds of kinetics. Incorporation into all the mitochondrial subfractions and the nuclear fraction rose gradually to a plateau value at about 20min., in contrast with that into the two microsomal fractions which rose rapidly to a peak value about seven times that for the mitochondrial fractions. The kinetics for the incorporation into mitochondrial cytochrome c showed a plateau value at 30min. about three times that for the total mitochondrial protein. There was no difference in the specific radioactivity of the mitochondrial cytochrome c extracted with water or 0.15m-sodium chloride or between the different mitochondrial subfractions. In contrast, the cytochrome c isolated from water extracts of the microsomal fractions had a lower specific radioactivity than that obtained from the 0.15m-sodium chloride extract. The specific radioactivity of the latter showed a rapid rise to a peak value about four times that for the mitochondrial cytochrome c, and the shape of the curve was similar to that for the total protein of the microsomal fraction. The results suggest that cytochrome c is synthesized in toto by the morphological components of the microsomal fraction. It seems first to be bound tightly to a microsomal particle, passing then to a looser microsomal binding and being finally transferred to the mitochondria. The newly synthesized cytochrome c in the mitochondrion could not be differentiated from the old by its degree of extractability at pH 4.0.     

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [³H]plamitate, [³H]oleate or [³H]arachidonate and [¹⁴C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total ³H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [¹⁴C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of ³H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [¹⁴C]glycerol labelling, generally the ¹⁴C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of ³H-labelled fatty acid/[¹⁴C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with ³H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.     

Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of microsome-catalyzed covalent binding in the target tissue

Using ³H-dioxane, the distribution of dioxane among a number of tissues and various subcellular fractions of rat liver was studied. At various times after i.p. injection, dioxane was found to distribute more or less uniformly among various tissues (liver, kidney, spleen, lung, colon and skeletal muscle), consistent with its polar/nonpolar nature. Studies of the nature of dioxane binding, however, revealed that the extent of “covalent” binding (as measured by incorporation into lipid-free, acid-insoluble tissue residues) was significantly higher in the liver (the main carcinogenesis target tissue), spleen and colon than that in other tissues. Investigations of the subcellular distribution in liver indicated that most of the radioactivity was in the cytosol, followed by the microsomal, mitochondrial and nuclear fractions. The binding of dioxane to the macromolecules in the cytosol was mainly noncovalent. The percent covalent binding was highest in the nuclear fraction, followed by mitochondrial and microsomal fractions and the whole homogenate. Pretreatment of rats with inducers of microsomal mixed-function oxidases had no significant effect on the covalent binding of dioxane to the various subcellular fractions of the liver. There was no microsome-catalyzed $\text{in}$$\text{vitro}$ binding of ³H- or ¹⁴C-dioxane to DNA under conditions which brought about substantial binding of ³H-benzo[a]pyrene.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Phospholipide Turnover in Microsomal Membranes of the Pancreas during Enzyme Secretion

After incubation of pigeon pancreas slices with P³² and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P³² incorporated into the phospholipides. The remaining phospholipide P and P³² were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P³² radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C¹⁴ in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P³². In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C¹⁴ in each phospholipide which showed a stimulation of P³² incorporation. The significance of the turnover of phosphatides in microsomal membranes in relation to the mechanism of secretion is discussed.     

Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae.

To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.     

Sub-cellular localization of radioactive steroids following administration of testosterone- 3 H in the androgen dependent mouse tumor, Shionogi carcinoma 115

The mouse androgen dependent tumor Shionogi carcinoma 115 or the non-responsive tumor Shionogi carcinoma 42 were transplanted into 40 mice and testosterone-³H (0.13 μg/20 μCi) was then injected into each animal. The retention of ether soluble radioactivity in the nuclear, mitochondrial, microsomal and cytosol fractions was higher in combined seminal vesicle-prostate tissue and in Shionogi carcinoma 115 than in Shionogi carcinoma 42, muscle, and spleen.     

Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.     

The use of zonal centrifugation to study membrane formation during the life cycle of mammalian cells. Synthesis of `marker'' enzymes and other components of cellular organelles

1. The activity of enzymes characteristic of microsomes (NADPH-cytochrome c reductase and uridine diphosphatase) and of inner mitochondrial membranes (cytochrome c oxidase and succinate-cytochrome c reductase) increases during the cell cycle of P815Y neoplastic mast cells in concert with total protein. The activity of glutamate dehydrogenase, an enzyme of the mitochondrial matrix, increases in a somewhat different manner. 2. The specific activity of mitochondrial structures involved in energy-coupling measured with a fluorescent probe remains constant during the cell cycle. 3. Mitochondrial and microsomal protein increases during the cycle at the same time as total protein; nuclear protein increases rather more sharply. 4. The rate of incorporation of labelled choline or inositol into nuclear, mitochondrial or microsomal phospholipid during the cell cycle follows the rate of incorporation into total phospholipid. 5. It is concluded that the major components of cellular membranes are synthesized, like total protein or phospholipid, throughout most of the intermitotic period.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

A cyto-chemical study on the pancreas of the guinea pig. III. In vivo incorporation of leucine-1-C14 into the proteins of cell fractions

DL-leucine-1-C(14) was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.