Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

Cytomatrix synthesis in MDCK epithelial cells

Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak (J. Biol. Chem., 256:4863-4870, 1981), was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with [14C]leucine over several days and then pulse-labeled for 4 hours with [3H]leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form. The results suggest that metabolic coupling between individual cellular filament systems is not strict. The data are, however, consistent with models that predict that assembly of a subcellular structure influences the turnover of its component proteins.     

Dynamics of extracellular DNA in the marine environment

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Dynamics of extracellular DNA in the marine environment.

The production and turnover of dissolved DNA in subtropical estuarine and oligotrophic oceanic environments were investigated. Actively growing heterotrophic bacterioplankton (i.e., those capable of [3H]thymidine incorporation) were found to produce dissolved DNA, presumably through the processes of death and lysis, grazing by bacteriovores, and excretion. Production of dissolved DNA as determined by [3H]thymidine incorporation was less than or equal to 4% of the ambient dissolved DNA concentration per day. In turnover studies, the addition of [3H]DNA (Escherichia coli chromosomal) to seawater resulted in rapid hydrolysis and uptake or radioactivity by microbial populations. DNA was hydrolyzed by both cell-associated and extracellular nucleases, in both estuarine and offshore environments. Kinetic analysis performed for a eutrophic estuary indicated a turnover time for dissolved DNA as short as 6.5 h. Microautoradiographic studies of bacterial populations in Tampa Bay indicated that filamentous and attached bacteria took up most of the radioactivity from [3H]DNA. Dissolved DNA is therefore a dynamic component of the dissolved organic matter in the marine environment, and bacterioplankton play a key role in the cycling of this material.     

Protein metabolism in preimplanted mouse ova

Intracellular specific radioactivity of an amino acid (leucine) was measured in preimplantation mouse ova. The measured specific activity along with leucine incorporation rates allow calculation of the protein synthetic rate in mouse ova. Unfertilized ova, fertilized ova, two-cell ova, and blastocysts convert amino acid to protein at the rate of 8.60, 7.36, 6.92, and 56.68 × 10^?13 moles of amino acid per hour per ovum, respectively. The specific activity measurements also allowed the calculation of intracellular leucine pool size. For the unfertilized, fertilized, two-cell, and blastocyst stage, the endogenous leucine pool was 11.9, 15.9, 6.4, and 62.4 × 10^?14 moles of leucine, respectively. Most of the increase in pool size in the blastocyst probably results from the increase in total volume of cells. Protein degradation measurements indicated a marked difference between protein turnover in the two-cell and blastocyst stage. Approximately 10% of the protein at the two-cell stage turns over with a half-life of 18.3 hr and 35% of the protein at the blastocyst stage turns over with a half-life of 11.2 hr. The large remaining fraction of protein turns over much more slowly.     

Organic Carbon Degradation in Arctic Marine Sediments,Svalbard: A Comparison of Initial and Terminal Steps

Degradation of marine organic matter under anoxic conditions involves microbial communities working in concert to remineralize complex substrates to CO ₂ . In order to investigate the coupling between the initial and terminal steps of this sequence in permanently cold sediments, rates of extracellular enzymatic hydrolysis and sulfate reduction were measured in parallel cores collected from 5 fjords on the west and northwest coast of Svalbard, in the high Arctic. Inventories of total dissolved carbohydrates were also measured in order to evaluate their potential role in carbon turnover. Polysaccharide hydrolysis rates exhibited substrate-related and, to a lesser extent, depth-related differences (p < 0.0001); laminarin hydrolysis was consistently most rapid at nearly all depths and sites, and fucoidan hydrolysis was least rapid. Although there was a high degree of variability in parallel cores, sulfate reduction rates also exhibited statistically significant depth-and station-related differences. A comparison with data from previous investigations in Svalbard sediments suggests that this variability is linked to substrate availability rather than to organism distribution. Total dissolved carbohydrate concentrations were comparable to those measured in more temperate sediments, and likely comprise a considerable fraction of porewater dissolved organic carbon. A comparison of dissolved carbohydrate inventories with hydrolysis and sulfate reduction rates suggests that the turnover of carbon through the dissolved pool occurs quite rapidly, on the order of a few days to weeks. The transformation of particulate to dissolved organic matter must also be sufficiently rapid to maintain the measured rates of terminal remineralization.     

Estimating protein turnover with a [15N,13C]leucine tracer: a study using simulated data.

We explore the use of [15N,13C]leucine tracer to estimate whole-body fractional rates of a fast-turning-over protein pool employing synthetic data. The kinetics of [15N,13C]leucine tracer are simplified compared with those of traditional leucine tracers and benefit from irreversible transamination to [13C]alpha-ketoisocaproaic acid (KIC) resulting in a simplified model structure. A three-compartment model of [15N,13C]leucine kinetics was proposed and evaluated using data generated by a Reference Model (based on a model by Cobelli et al.). The results suggest that fractional turnover rates of a fast-turning-over protein pool can be estimated with a low but acceptable precision during a six-hour constant intravenous infusion of [15N,13C]leucine with frequent sampling of plasma tracer-to-tracee ratio (TTR) of [15N,13C]leucine. We conclude that [15N,13C]leucine may be useful for the measurement of protein kinetics and its full potential should be explored in clinical studies with compartmental data analysis.     

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.     

Epidural blockade improves substrate utilization after surgery

The purpose of this study was to test the hypothesis that epidural blockade with local anesthetic improves the anticatabolic effects of glucose after colorectal surgery. Sixteen patients were randomly assigned to undergo a 6-h stable isotope infusion study (3 h fasted, 3 h glucose infusion at 4 mg. kg(-1). min(-1)) on the second postoperative day with or without perioperative epidural blockade. Protein synthesis, breakdown and oxidation, and glucose production and clearance were assessed by L-[1-(13)C]leucine and [6, 6-(2)H(2)]glucose. Epidural blockade did not affect protein and glucose metabolism in the fasted state. Glucose infusion increased glucose clearance (P < 0.05), accompanied by an increase in the respiratory quotient (P < 0.05) and a decrease in leucine oxidation (P < 0.05) only in the presence of epidural blockade. An inverse correlation (r = -0.74, P < 0.05) between changes in glucose clearance and leucine oxidation was observed. In conclusion, epidural blockade facilitates whole body glucose uptake and inhibits endogenous protein oxidation after abdominal surgery, indicating a shift from a protein to a more glucose-dominated substrate utilization.     

Use of the [(14)C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton.

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [(14)C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 microM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 microM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.     

Metabolic properties of substrate-attached glycoproteins from normal and virus-transformed cells.

Balb/c 3T3, SV40-transformed 3T3 (SVT2), and Con A revertant variants of transformed cells leave a layer of glycoprotein on the culture substrate upon EGTA mediated removal of cells. The metabolic properties of this substrate-attached material (glycoprotein) have been examined. Pulse and cumulative radiolabeling experiments with glucosamine and leucine precursors established that this substrate-attached material accumulates on the substrate in growing cultures until cells have completely covered the substrate. The synthesis and/or deposition of the material diminished dramatically in cultures whose substrates had been completely covered with cells as observed microscopically, even though the contact-inhibited cell lines continued to make cell-associated and medium-secreted glycoproteins and transformed cells continued to divide and form multilayered cultures. Pulse-chase analysis using long periods of pulsing with radioactive leucine demonstrated that these glycoproteins are deposited directly on the substrate by cells and not subsequent to secretion into the medium. The substrate-attached material accumulated during long pulses was stably adherent to the substrate and displayed little appreciable turnover during 3 days of chasing of either sparse or dense cultures. Short-term pulse-chase analysis with leucine revealed two metabolically different pools of material-one which turns over very rapidly with a half-life of 2-3 hr (observed in both low-density and high-density cultures) and a second pool which is stably deposited on the substrate and whose proportion increased with the length of the radiolabeling period. No appreciable differences in the metabolic properties of substrate-attached material were observed in the three cell types studied during growth on a plastic substrate. These results are discussed with regard to the implicated roles of these glycoproteins in in mediating adhesion of normal and virus-transformed cells to the substrate.     

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.     

Disruption of planktonic phosphorus cycling by ultraviolet radiation

Ultraviolet radiation (UVR) may alter phosphorous (P) cycling by plankton through changes in the acquisition and/or regeneration of dissolved P. However, to date an effect of UVR on the uptake of P has not been observed at ambient phosphate (PO₄ ³⁻) concentrations. This has lead to the conclusion that the uptake of P by plankton may be insensitive to UVR. Past research has been limited to a few individual systems, prolonged incubations in bags, or lab cultures. We suspect that experimentation with natural plankton assemblages across broader environmental and/or chemical gradients is required to appreciably understand how UVR may alter P kinetics. Therefore, our study aimed to determine the effect of UVR on the turnover time of the dissolved PO₄ ³⁻ pool, the regeneration of dissolved P, the turnover rate of particulate P, and on PO₄ ³⁻ concentrations in natural plankton assemblages across broad environmental and chemical gradients. Second we aimed to assess how UVR may alter phosphatase activity and, determine if a change in phosphatase activity under UVR irradiance is correlated with a change in P uptake as proposed in the literature. Studies were conducted on 18 thermally stratified or polymictic lakes located in Ontario and Saskatchewan, Canada. Lake water samples were exposed to one of three experimental treatments: control, photosynthetically active radiation (PAR), or photosynthetically active radiation plus ultraviolet radiation (PAR + UVR). Our study is the first to demonstrate that UVR exposure has the potential to alter P cycling at ambient (picomolar) PO₄ ³⁻ concentrations. We have demonstrated that the turnover time of the PO₄ ³⁻ pool increases under UVR irradiance (i.e., P uptake decreases), while the regeneration rate of dissolved P and turnover rate of planktonic P are generally not affected; with the net effect being an increase in steady state PO₄ ³⁻ concentration (ssPO₄ ³⁻). Alkaline phosphatase activity (APA) in the dissolved and particulate fractions was significantly reduced in PAR + UVR treatments, but unrelated to changes in P uptake. In summary, we have demonstrated that the cycling of P may be disrupted by UVR, with a decrease in the uptake of P and the accumulation of PO₄ ³⁻ in the dissolved pool. This, in turn may exacerbate planktonic P limitation, alter the nutrient stoichiometry of plankton and/or indirectly alter rates of primary production in limnetic systems.     

DNA SYNTHESIS IN RANA PIPIENS TADPOLE LIVER DURING TRIIODOTHYRONINE-INDUCED METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during Ts-induced metamorphosis of R. pipiens liver was investigated. Rates of DNA synthesis were estimated by rates of 3 H-thymidine incorporation into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. During T3 -induced metamorphosis, periods of DNA synthesis and fluctuations in DNA content preceded expression of biochemical differentiation as measured by the enzyme arginase, and fluctuations in synthesis rates preceded corresponding fluctuations in content. The earliest response to T3- , was a 50% decrease in liver DNA, followed by increases in thymidine incorporation at 16 hr, 2 days, and 5-8 days. The size of the endogenous thymine pool was not significantly altered by T3 These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthetic rates and content during metamorphosis. Expression of thyroxin-induced development of the tadpole liver appears to be associated with both proliferation and cellular death, and metamorphosis of the liver cannot be occurring in a “fixed population of cells.”     

Whole body and tissue cholesterol turnover in the baboon

Cholesterol turnover was studied in four baboons by injecting [14C]cholesterol 186 days and [3H]cholesterol 4 days before necropsy, and fitting a two- or three-pool model to the resulting specific activity-time data. At necropsy, cholesterol mass and specific activity were determined for the total body (minus the central nervous system) and for many tissues. A pool model permits the estimation, from the plasma specific activity-time curve alone, of total body cholesterol within a limited range, depending upon the extent of side pool synthesis. The principal aim of this study was to estimate the extent of cholesterol synthesis in the side pools of the model, by computing the amount of side pool synthesis needed to equal the measured total body cholesterol. Central pool synthesis varied from 61 to 89% of the total cholesterol production rate. Thus, approximately 25% (11 to 39%) of the production rate arose from peripheral (pool 3 for the three-pool, and pool 2 for the two-pool model) cholesterol synthesis. Moreover, the finding that the measured total body cholesterol fell within the range obtained from the kinetic analysis by using reasonable assumptions (namely, that zero or that half the production rate occurred in the side pools), provides evidence for the physiological validity of the model. A second aim of this study was to explore cholesterol turnover in various tissues. A pool model predicts that rapidly turning over tissues will have higher specific activities at early times and lower specific activities at later times after injection of tracer relative to slowly turning over tissues, except where significant synthesis occurs. Tissues were ranked 1 to 17 for 3H and 17 to 1 for 14C cholesterol specific activity values. Except for the GI tract and testis, the tissues had similar ranks for both 3H and 14C, further validating model predictions. Results in all four baboons were similar. Turnover rates for the different tissues loosely fell into three groups which were turning over at fast, intermediate, and slow rates. Finally, the magnitude of variation of cholesterol specific activity was moderate for several distributed tissues (fat, muscle, arteries, and the alimentary tract), but was small for liver. Cholesterol turnover in serial biopsies of skin, muscle, and fat could, however, be fitted with a single pool to estimate tissue turnover rates.     

Indices of mitotic cell division in sebaceous glands]

The main indices of mitotic cell division in rat sebaceous glands (external auditory meatus and tarsales gl.) were studied autoradiographically using H3-thymidine and with colchicine method. The duration of mitotic cycle and its separate phases, the number of cells involved in the proliferative pool, as well as the turnover of terminals of the epithelium in both the glands were stated to be nearly identical. The duration of the mitotic cycle was: T -- 28.1 hour; tG1 -- 18.64; tS -- 6.3; tG2 -- 1.80; tM -- 1.34 hours. The proliferative pool (Pc) -- 31.45%, turnover of the basal layer cells -- 89.25 hours. These indices for the stratified epithelium of excretory ducts were respectively; T -- 33.0 hours; tG1 -- 21.74; --8.06; tG2 -- 1.6; tM -- 1.6; Pc -- 26.8% and the turnover for the cells of the basal layer -- 123 hours. Thus, the sebaceous glands are to be regarded as organs where a rapid renovation of epithelia cells occurs.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.     

Use of the [14C]Leucine Incorporation Technique To Measure Bacterial Production in River Sediments and the Epiphyton

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [¹⁴C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 μM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 μM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.