Huntington''s-chorea fibroblasts. Cellular protein glycosylation.

Five cell cultures of Huntington's-chorea fibroblasts exhibit greater than normal protein and lipid glycosylation when labelled with [U-14C]glucosamine. Oligosaccharide--polypeptide chains from all molecular-weight ranges are labelled differentially on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This difference in protein glycosylation is not accompanied by any apparent difference in general cellular protein synthesis or by a differential rate of glucosamine uptake or decreased degradation of [14C]glycosylated macromolecules. Additionally [U-14C]glucosamine exclusively labels hexosamines and sialic acid of cellular macromolecules.     

Degradation of zinc-metallothionein in monolayer cultures of rat hepatocytes

The degradation of zinc-metallothionein (MT) was studied in monolayer cultures of adult rat hepatocytes. Hepatocytes were incubated overnight in serum-free medium containing either [35S]cysteine or [3H]leucine and 100 microM zinc to induce MT synthesis. Total cellular 35S-MT was measured in the heat-stable extract of cell homogenate and quantified by fast protein liquid chromatography. When zinc was removed from the medium, 35S-MT turnover was almost 3-fold faster than that of [3H]Leu protein (t1/2 = 11 and 29 hr, respectively). The decrease in the cellular level of 35S-MT reflected degradation since less than 1% of total cellular 35S-MT was secreted into the medium. The rate of MT degradation was inversely proportional to cellular zinc content. In contrast, the degradation of [3H]Leu protein was not affected by changes in cellular zinc concentration. Chloroquine, a lysosomotrophic amine, and tosyl lysine chloromethyl ketone, an inhibitor of trypsin-like neutral protease activity, inhibited 35S-MT degradation by 65% and 50%, respectively, when cells were incubated in medium with 1 microM zinc. Turnover of [3H]Leu protein, but not 35S-MT, was enhanced by insulin deprivation. These data suggest that the degradation of hepatic MT (i) is primarily regulated by cellular zinc content and (ii) occurs in both lysosomal and nonlysosomal compartments.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

High-Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.     

A low-molecular-weight inhibitor of the neutral proteinase from rat intestinal smooth muscle.

1. Rat intestinal smooth muscle was shown to contain endogenous inhibitory activity towards the neutral trypsin-like muscle proteinase described previously [Beynon & Kay (1978) Biochem. J. 173, 291--298]. 2. Comtamination of the muscle tissue by mucosal, blood and pancreatic inhibitors was shown to be unlikely. 3. The inhibitory activity was resolved into high- and low-molecular-weight components. 4. The low-molecular-weight component was purified to homogeneity. It has a molecular weight of approx. 9000 and was stable over the pH range 3--11. 5. It inhibited the muscle proteinase competitively (Ki congruent to t microM), but had no effect on any of the other proteinases tested. 6. Leupeptin also inhibited the muscle proteinase competitively (Ki congruent to 0.3 microM), whereas the low-molecular weight proteins gastrin, glucagon and insulin B-chain had very little effect. 7. A role for a weakly binding inhibitor in modulating the influence of the neutral proteinase on intracellular protein degradation is considered.     

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.     

Characteristics of intracellular metabolism of procollagen and other proteins in human embryo fibroblasts in trisomy for chromosome 7

A comparative study of the relative rates of intracellular total protein metabolism in diploid and aneuploid (with trisomy for chromosome 7) human embryo fibroblasts in the logarithmic and stationary growth phases was carried out. Using double labeling with [14C]proline (24 hrs) and [3H]proline (3 hrs), it was found that: the rates of intracellular protein metabolism during transition to the stationary phase of growth are increased in diploid cells and decreased in cells with trisomy for chromosome 7; the relative rate of protein metabolism in the logarithmic phase is higher in trisomic cells than in diploid ones. The intracellular degradation of procollagen in trisomic cells is increased approximately by 17% as compared to normal fibroblasts. Treatment of cell lysates with bacterial collagenase revealed the presence of procollagen incomplete degradation products in anomalous fibroblasts. The observed differences in the rates and mode of protein metabolism during transition of diploid and trisomic fibroblasts to the stationary phase of growth suggest that the odd autosome interferes with the normal coordinated activity of genes in chromosomes.     

Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells

The rates of [³H]N^τ-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [³H]methionine, were used to measure myofibrillar protein degradation. In fused C₂C₁₂ myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions ± leucine, but in the absence of [³H]-methionine, rates of protein accretion and synthesis over 24–48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types. © 1996 Wiley-Liss, Inc.     

Effects of beta-lapachone, a peroxide-generating quinone, on macromolecule synthesis and degradation in Trypanosoma cruzi

Incubation of Trypanosoma cruzi epimastigotes with beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), a lipophilic o-quinone, produced inhibition of [3H]thymidine, [3H]uridine, and L-[3H]leucine incorporation into DNA, RNA, and protein, respectively. With 1.6 microM beta-lapachone, DNA synthesis was preferentially inhibited. The inhibition was irreversible, and time and concentration dependent. Other effects of beta-lapachone were (a) inhibition of 3H precursor uptake into epimastigotes, (b) exaggerated degradation of DNA, RNA, and protein, (c) increased unscheduled synthesis of DNA, and (d) increased number of strand breaks in nuclear and kinetoplast DNA. DNA damage by 1.6 microM beta-lapachone was repaired by reincubating the drug-treated epimastigotes in fresh medium for 24 h, but with 7.8 microM beta-lapachone DNA damage was irreversible. The p-quinone isomer alpha-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione), was less effective than beta-lapachone, especially on DNA and RNA synthesis, and did not stimulate unscheduled DNA synthesis. Since beta-lapachone redox cycling in T. cruzi generates oxygen radicals while alpha-lapachone does not (A. Boveris, R. Docampo, J. F. Turrens, and A. O. M. Stoppani (1978) Biochem. J. 175, 431-439), the summarized results support the hypothesis that oxygen radicals contribute to beta-lapachone toxicity in T. cruzi.     

Regulation of protein turnover by recombinant human insulin-like growth factor-I in L6 myotube cultures

Muscle cell culture experiments were conducted to determine the relative regulatory effects of insulin-like growth factors (IGF) on protein turnover. The effects of recombinant (rc) human IGF-I, ovine somatomedin (oSm/oIGF-I), and insulin on rates of protein labeling and degradation in L6 myotube cultures were evaluated. Myotube cultures were treated with growth factors following a 4-h serum-free incubation period. Protein labeling was measured by determining the rate of [3H] leucine incorporation into cell protein. Protein degradation was measured by a pulse-chase procedure using [3H] leucine. The apparent half maximal stimulation of protein labeling (12%, 8%, 7%) occurred at approximately .1 nM rcIGF-I, 1 nM oSm/oIGF-I and 15 nM insulin, respectively. The apparent half maximal inhibition of proteolysis (18%, 15% and 11%) occurred at .4 nM rcIGF-I, .6 nM oSm/oIGF-I and 4 nM insulin, respectively. The magnitude of the response for protein labeling and degradation was greatest for rcIGF-I. The results provide additional evidence that IGFs play a primary role in regulating protein turnover in muscle.     

The long-term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts.

The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20-min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol-induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine-prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40-80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50-200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA-induced formation of ethanolamine, catalyzed by a phospholipase-D-type enzyme. In membranes isolated from TPA+ethanol-treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down-regulation of PKC by prolonged (24-h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of alpha-PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 microM) to fibroblasts inhibited TPA+ethanol-induced formation of ethanolamine phosphate. These results suggest that alpha-PKC-mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.     

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl-L-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 +/- 1.4% to 10.5 +/- 2.4% with 10 microM TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 microM TLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 micrograms/ml leupeptin or 60 micrograms/ml pepstatin A. However, the production of collagen was reduced to 10.9 +/- 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 microM TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.     

DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.     

Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins.

Tyrphostins, which block protein tyrosine kinase activity, were studied for their inhibitory action on platelet-derived growth factor (PDGF)-induced proliferation of human bone marrow fibroblasts. Of the seven tryphostins examined, tyrphostin AG370 was found to be the most potent blocker against PDGF-induced mitogenesis (IC50 = 20 microM). This PTK blocker also blocks mitogenesis induced by epidermal growth factor (IC50 = 50 microM) and human serum (IC50 = 50 microM), but with lower efficacy. In digitonin-permeabilized fibroblasts as well as in intact fibroblasts, tyrphostin AG370 inhibits PDGF receptor autophosphorylation and the tyrosine phosphorylation of intracellular protein substrates (pp120, pp85, and pp75) which coprecipitate with the PDGF receptor. In comparison to AG370, AG18, a potent EGF receptor blocker, was less efficient in inhibiting PDGF-induced proliferation of fibroblasts and phosphorylation of the intracellular protein substrates. Under the conditions in which AG370 inhibits PDGF-induced mitogenesis and phosphorylation, it does not affect [125I]PDGF internalization and enhance [125I]PDGF binding. These findings suggest that AG370, which is an indole tyrphostin, may serve as a model for developing analogues with a therapeutic potential for treatment of diseases which involve abnormal cellular proliferation induced by PDGF.     

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reappraisal of the quantitative importance of non-skeletal-muscle source of N tau-methylhistidine in urine.

The claim of Millward, Bates, Grimble, Brown, Nathan & Rennie [(1980) Biochem. J. 190. 225--228] that muscle actomyosin contributes as little as 25% of urinary N tau-methylhistidine is not consistent with other published data from that group [Bates, DeCoster, Grimble, Holloszy, Millward & Rennie (1980) J. Physiol. (London) 303, 41 P] or with literature values. It appears likely that the turnover rate of muscle actomyosin has been considerably underestimated and that when realistic rates of protein turnover are used, muscle tissue remains the major contributor of N tau-methylhistidine in urine.     

High stearoyl-CoA desaturase protein and activity levels in simian virus 40 transformed-human lung fibroblasts

The precise role of monounsaturated fatty acid (MUFA) synthesis in cell proliferation and programmed cell death remains unknown. The strong correlation of high levels of MUFA and neoplastic phenotype suggest that the regulation of stearoyl CoA desaturase (SCD) must play a significant role in cancer development. In this study, the levels of SCD protein and activity were investigated in normal (WI38) and SV40-transformed (SV40-WI38) human lung fibroblasts. Thus, the activity of SCD on exogenous [14C]stearic acid and endogenous [14C]acetate-labeled fatty acids was increased by 2.2- and 2.6-fold, respectively, in SV40-WI38 compared to WI38 fibroblasts. Concomitantly, a 3.3-fold increase in SCD protein content was observed in SV40-transformed cells. Cell transformation also led to high levels of MUFA, which was paralleled by a more fluid membrane environment. Furthermore, the levels of PPAR-gamma, a well-known activator of SCD expression, were highly increased in SV40-transformed fibroblasts. SCD activity appeared linked to the events of programmed cell death, since incubations with 40 microM etoposide induced apoptosis in SV40 cells, and led to a decrease in fatty acid synthesis, SCD activity and in MUFA cellular levels. Taken together, these results suggest that SCD protein and activity levels are associated with the events of neoplastic cell transformation and programmed cell death.     

Regulation of the functional expression of hexose transporter GLUT-1 by glucose in murine fibroblasts: role of lysosomal degradation.

The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [3H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 microM) and ammonium chloride (10 mM) cause accumulation (both approximately 4-fold) of GLUT-1 protein and a small increase (both approximately 25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 microM) causes accumulation (approximately 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (less than or equal to 5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (approximately 4-fold) and GLUT-1 content (approximately 7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein degradation in rat liver during post-natal development.

Protein degradation rates for liver subcellular and submitochondrial fractions from neonatal (8-day), weanling (25-day) and adult rats were estimated by the double-isotope method with NaH14CO3 and [3H] arginine as the radiolabelled precursors [Dice, Walker, Byrne & Cardiel (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097]. Decreased protein degradation rates were found during post-natal development for homogenate, nuclear, mitochondrial, lysosomal and microsomal proteins. A decrease in degradation rates for the immunoisolated subunits of monoamine oxidase and pyruvate dehydrogenase was also observed in neonatal and weanling rats respectively. The results suggest coordinate degradation of the subunits of the multi-subunit enzyme pyruvate dehydrogenase. Pyruvate dehydrogenase has a faster rate of degradation in adult rat liver than does cytochrome oxidase. Data analysis suggests heterogeneity of protein degradation rates in the mitochondrial outer membrane and intermembrane space fractions at each developmental stage but not in the mitochondrial inner membrane or matrix fractions. Results obtained for protein degradation rates in adult rat liver by the method of Burgess, Walker & Mayer [(1978) Biochem. J. 176, 919-926] in general confirmed the results obtained for the adult rat liver by the above method. No evidence of a subunit-size relationship for protein degradation was found for proteins in any subcellular or submitochondrial fraction.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.