Amino acid analogues: uptake, pool formation and incorporation of phenylalanine and two halogenated derivatives in cultured mammalian cells.

HeLa cells take up Phe and two of its ring halogenated derivatives (pFPhe and pClPhe) with rpaidity, concentrating them against the external medium both at 4 and 37 degrees C. The majority of amino acid (greater than 90%) is accumulated without energy expenditures at 4 degrees C, and can be quickly discharged by normal cell washing procedures in saline. At 37 degrees C the freely-diffusible (FDP) pool is accompanied by another which develops more slowly and cannot diffuse out freely during washings with saline but is extractable with trichloracetic acid (the slowly-diffusible pool, SDP, or more conventionally, the acid-soluble pool). Both of the analogues produced larger pools of the latter type than Phe itself from external concentrations ranging from 10(-5) to 10(-3) M. The incorporation of pFPhe into proteins over these same concentrations ranged from 30 to 90--95% of Phe incorporation, whereas pClPhe showed negligible incorporation. From these and similar analyses it can be concluded that amino acid pools form largely independently of protein synthesis, but bear a close relationship with the external amino acid concentration. The fraction of total uptake into cellular pools entering the SDP was relatively constant over a wide range of external concentrations. pFPhe incorporation into cellular proteins produced the same labelling distribution of Phe. It appears to ener all proteins, the vast majority of which have similar half-lives and turnover rates to Phe proteins. In competition, little or no interference was experienced between the analogue and Phe in uptake and pool formation until excessive amounts of one or the other were present (50--100x). By contrast, incorporation of pFPhe into protein was markedly reduced by the presence of Phe. However, the development of normal or large pools of pFPhe or Phe in cells prior to 3H-Phe incorporation did not affect the linear incorporation pattern of the radioisotope into protein. The relationship of pools to protein synthesis is discussed, and it is concluded that, although the SDP could contain potential precursor molecules for protein synthesis, it does not usually act as the direct supplier of amino acid for protein synthesis. Alternative explanations for precursor supply are discussed.     

Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) [3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.     

Functional homogeneity of leucine pool in retina cells

Data on leucine metabolism in isolated rabbit retina are examined for evidence, for or against, a common intracellular pool of free leucine. Data include values for: concentrations, transport rates, degradative metabolism and protein incorporation of labelled leucine measured over a wide range of concentrations; protein incorporation of labelled threonine, measured simultaneously; and an indirect measurement of protein breakdown. The fall in labelled leucine incorporation into protein, when medium leucine was reduced below 100 microM, corresponded closely with the fall in intracellular specific activity predicted from rate of influx of labelled leucine from medium and rate of release of unlabelled leucine from protein breakdown. Protein incorporation of labelled leucine competed with decarboxylation and outward transport and reduced the free intracellular leucine in about the amounts predicted for a common pool. Implications for measurements using labelled amino acid are discussed.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

Protein synthesis in central nervous tissue: studies on retina in vitro

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.     

Amino acid pool and protein turnover during differentiation (spherulation) ofPhysarum polycephalum

The composition of the amino acid pool during spherulation was determined. It changes in size and in composition, the concentration of each amino acid behaving individually. The first response to the onset of spherulation either by starvation or osmotic shock (0.5 M mannitol) always is a decrease of the pool's size, which during further starvation expands for a short period and then decreases again. During development induces by mannitol in the presence of external amino acids, the pool size increases continuously after the initial depletion.As shown by radioactive labeling, amino acids were actively released from the plasmodium into a medium containing amino acids, but retained by the microplasmodia in an amino acid-free medium. The kinetics of the uptake of radioactive amino acids from the medium is biphasic, indicating the existence of multiple pools. Even after a labeling period of 8 h the amino acid pool is not yet in equilibrium with the medium. The possibility of a compartimentation of the pool was confirmed by density labeling of two different enzymes.Whereas the turnover of total protein is only very low during growth, it is rather high in spherulating microplasmodia. At least 70% of the originally existing protein is degraded during this development, while, simultaneously, at least 50% of the protein present after 24 h starvation is newly synthesized during that period.     

Kinetics of proteoheparan sulfate synthesis, secretion, endocytosis, and catabolism by a hepatocyte cell line

The metabolism of heparan sulfate proteoglycan was studied in monolayer cultures of a rat hepatocyte cell line. Late log cells were labeled with 35SO4(2-) or [3H] glucosamine, and labeled heparan sulfate, measured as nitrous acid-susceptible product, was assayed in the culture medium, the pericellular matrix, and the intracellular pools. Heparan sulfate in the culture medium and the intracellular pools increased linearly with time, while that in the matrix reached a steady-state level after a 10-h labeling period. When pulse-labeled cells were incubated in unlabeled medium, a small fraction of the intracellular pool was released rapidly into the culture medium while the matrix heparan sulfate was taken up by the cells, and the resulting intracellular pool was rapidly catabolized. The structures of the heparan sulfate chains in the three pools were very similar. Both the culture medium pool and the cell-associated fraction of heparan sulfate contained proteoheparan sulfate plus a polydisperse mixture of heparan chains which were attached to little, if any, protein. Pulse-chase data suggested that the free heparan sulfate chains were formed as a result of catabolism of the proteoglycan. When NH4Cl, added to inhibit lysosomal function, was present during either a labeling period or a chase period, the total catabolism of the heparan sulfate chains to monosaccharides plus free SO2-4 was blocked, but the conversion of the proteoglycan to free heparan sulfate chains continued at a reduced rate.     

Involvement of cyclic AMP and calcium in exocrine protein secretion induced by vasoactive intestinal polypeptide in rat parotid cells

The effects of vasoactive intestinal polypeptide (VIP) on exocrine protein secretion were studied in enzymatically dispersed cell aggregates from rat parotid glands. VIP (10(-9) - 10(-7) M) stimulated secretion of alpha-amylase in a dose-dependent manner. The VIP-induced release of alpha-amylase was potentiated in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP of the dispersed cells were increased 6.7-fold after stimulation for 10 min by VIP (10(-7) M). The VIP-induced release of alpha-amylase was reduced by 40% when cells were incubated in a Ca2+-free medium in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Efflux of 45Ca2+ was significantly increased over basal levels by stimulation with VIP (10(-8) and 10(-7) M), but this increased efflux was approximately only half the increased efflux induced by carbachol (10(-5) M). VIP had no effect on the incorporation of [14C]leucine into protein by parotid cells, whereas incorporation was reduced to 30% of the control value by carbachol (10(-5) M). Thus, the VIP-ergic secretory response in the rat parotid gland is associated with a raised intracellular cyclic AMP level and the mobilisation of a different intracellular Ca2+ pool than that mobilised by carbachol. It is, therefore, closely analogous to the beta-adrenergic response.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae.

By using the Cu2+ method (Y. Ohsumi, K. Kitamoto, and Y. Anraku, J. Bacteriol. 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined. Histidine and lysine in the medium expanded the vacuolar pool extremely. Glutamate also accumulated in the cells, but mainly in the cytosol. The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool. Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM. This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation. Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate. The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.     

Intracellular amino acids and protein synthesis in Hela cells

1. When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein.
2. Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected.

From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.     

Heterotrophic Activity of Deep-Sea Sediment Bacteria

Sediment samples, containing mixed microbial populations that were decompressed during retrieval from 7,750 and 8,130 m in the Puerto Rican Trench, were recompressed and incubated at the approximate in situ temperature (3 C) and pressure (775 or 815 atm) in the presence of 14C-labeled amino acids. Heterotrophic activity (total uptake, CO2 respiration, and cellular assimilation) and cellular-associated "pool" concentrations were measured. Compared with atmospheric controls held at 3 C, the total uptake at elevated pressure at 3 C was reduced, on an average, 55 times, CO2 respiration was reduced 45 times, and cellular assimilation was reduced 69 times. Rate of total uptake at elevated pressure was found to range from 4.0 X 10(-11) mug/cell per h for leucine to 2.61 X 10(-10) mug/cell per h for an amino acid mixture. Also, the percentage of total uptake at elevated pressures, respired as CO2, increased at the expense of cellular assimilation (ca. 22% increase). Two cellular-associated amino acid pools were detected, a large, loosely bound, outer pool and a small, tightly bound internal pool. The loosely bound outer pool was removed by a change in the pH of the incubation medium. Even though heterotrophic uptake and the outer, cellular-associated pool were markedly reduced at an elevated pressure, the percentage of total uptake calculated for the unincorporated, tightly bound, intracellular pool was 2 to 19 times that obtained for cultures held at 1 atm. The results were interpreted as indicating that bacterial metabolism and biosynthesis in the deep sea are markedly reduced, with a greater proportion of metabolic activity devoted to cellular maintenance.     

Use of silicone fluids in studies of cellular amino acids

In numerous cellular studies, cells labeled with radioisotopes have been separated from the labeling medium by an aqueous solution in order to determine the quantity of internalized labels; however, the aqueous wash tends to remove significant labeling from the cells. Therefore, in order to preserve all of the internalized labels, non-aqueous medium such as silicone fluids may be used. The termination of the labeling is achieved in the silicone method when, upon centrifugation, the cells separate from the medium and enter the silicone fluid to sediment to the tube bottom. This sedimentation of cells placed above a layer of silicone fluid exhibits a critical dependence on the centrifugal force, and gives rise to an uncertainty of only 2 s in determining the time of separation of cells from the medium using General Electric F-50 silicone fluid and a modified Beckman J2-21 centrifuge. It is therefore possible to determine the kinetics of incorporation of labeled amino acids into intracellular pools and proteins. In particular, since this silicone wash method determines the size of the total pool and the aqueous wash method determines the size of the acid-extractable pool, the simultaneous measurements of the size of both pools leads to the determination of the kinetics of labeling of the free amino acid pool. Among many possible applications and extensions of these methods, the studies of formation of intracellular pools and relations among different pools of transported molecules, such as water and amino acids, appear promising.     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.     

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.     

Regulation of secretion of parathormone and secretory protein-I from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)- isoproterenol

Dispersed porcine parathyroid cells were incubated at calcium concentrations between 0.5 and 3.0 mM in the presence of 3H- or 14C- amino acids to label newly synthesized parathormone. Up to four times more hormone was secreted at the lower calcium concentration but its specific radioactivity, from 30 to 50 times that of the intracellular pool, did not change. Dibutyrl cyclic AMP doubled immunoactive parathormone secretion at each calcium concentration, but there was no increase in secretion of radioactive hormone if labeled amino acids and secretagogue were added simultaneously. Similarly, when the intracellular pool of parathormone was prelabeled with 3H-amino acids and then the cells were incubated in 14C-amino acids and dibutyryl cyclic AMP, the entire increase in hormone secreted was derived from the prelabeled pool. (1)-isoproterenol increased intracellular cyclic AMP and acted on hormone secretion in a manner indistinguishable from dibutyryl cyclic AMP. In similar double-label experiments dibutyryl cyclic AMP preferentially enhanced secretion of secretory protein-I, a calcium-regulated protein of the parathyroid of unknown function. Calcium, alone, inhibited the intracellular level of cyclic AMP in a concentration-dependent fashion. These data are consistent with the existence in the parathyroid cell preparation of two hormone and secretory protein pools that may be individually recruitable--one consisting of most recently synthesized protein, the other consisting of older "storage" protein. The data do not allow one to decide whether the two pools coexist within individual cells or whether, instead, they exist in separate cells of the dispersed gland preparation.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1. The accumulation of [1-(14)C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1-(14)C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.     

Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.     

Tunicamycin-treated rat heart cell cultures synthesize an inactive nonreleasable lipoprotein lipase

Cells isolated from newborn rat hearts were cultured in the presence of 100 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Lipoprotein lipase activity was present in an intracellular and heparin-releasable pool and was also secreted into the culture medium. Treatment of the cultures with 5 micrograms/ml tunicamycin caused almost complete loss of lipoprotein lipase activity in all three compartments. In control cultures, immunoblotting of lipoprotein lipase derived from all three pools revealed a single band of lipoprotein lipase with an apparent Mr of 56,000. In the tunicamycin-treated cultures, the enzyme appeared only intracellularly and had an apparent Mr of 49,000. No immunoreactive enzyme was found in the medium. Thus, glycosylation of lipoprotein lipase in heart cell cultures is mandatory for enzyme activity and translocation from an intracellular to the heparin-releasable pool and for secretion into the medium.     

The catabolism of plasma glycoproteins in normal and injured rats

The catabolism of (14)C-labelled plasma glycoprotein in rats was studied after injecting homologous plasma protein labelled in the N-acetylglucosamine and sialic acid moieties. In normal animals the catabolism was approximately described by a four-compartment model. The fractional rate of catabolism of the plasma-protein amino sugar was found to be 0.0305hr.(-1), corresponding to the degradation of 2.75mumoles/hr. The (14)C label was eliminated from the animals largely as carbon dioxide with a small proportion appearing in the urine. Freely circulating amino sugars or glycopeptides did not appear in the plasma as a result of the catabolic processes, and there was no evidence that the protein-bound amino sugars were reutilized in biosynthetic processes. A study of the distribution of (14)C label in the carcasses of animals 24hr. after injection provided evidence that the gastrointestinal tract accounted for 25-38% of the total catabolic pool; the lungs, kidneys, spleen and liver also appeared to contribute to catabolism. Studies were conducted with rats that had been treated with turpentine to induce an inflammatory reaction; the results could not be analysed kinetically, since the metabolism of plasma proteins in these animals did not appear to be in a steady state. The injected plasma protein disappeared from the intravascular pool more quickly than in normal animals, but there were no significant differences in the rates of excretion of the (14)C label.