Synthesis of macromolecules by the epithelial surfaces of Schistosoma mansoni: an autoradiographic study.

The use of tritiated leucine as a marker for protein synthesis and of tritiated glucosamine as a marker for polysaccharide/glycoprotein synthesis, is described. Adult worms were pulse-labelled by incubation in medium containing the substrate. Labelled worms were then incubated in chase medium, without labelled substrate, for varying lengths of time before fixation. The distribution of label which had been incorporated into macromolecules in the worm tissues, was examined by light and electron microscope autoradiography. It was estimated that the tegument and tegument cell bodies were the source of 67--80%, and the gut epithelium of 20--30%, of exportable leucine-containing protein. Conversely, the gut epithelium was the source of 72%, and the tegument cells 28%, of exportable glucosamine-containing polysaccharide. The specific activity of labelled protein reached a peak in the tegument cytoplasm after 1.5 h of chase incubation. Half of the labelled protein was secreted into the worm's environment by 3 h of chase incubation. The half-life of secretory protein in gut cells appears to be around 2 h. Labelled protein disappears from the gut lumen relatively rapidly but labelled polysaccharide remains in the lumen at high specific activity for at least 24 h. The major carbohydrate labelled may be the glycocalyx on the luminal surface of the gut epithelial cells. The results suggest that the bulk of worm secretions have a rapid turnover with a half-life of a few hours. Against this background of rapid mass secretion a slower process of membrane turnover would be difficult to detect and quantitatively small.     

Kinetic pulse-chase labeling study of the glucocorticoid receptor in mouse lymphoma cells. Effect of glucocorticoid and antiglucocorticoid hormones on intracellular receptor half-life

A kinetic pulse-chase labeling technique was used to measure the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine for 30 min and then cultured in the presence of unlabeled methionine (chase). Labeled receptors were quantitated at periodic time points during the chase by immunoadsorption to protein A-Sepharose using the BuGR2 monoclonal antireceptor antibody. The decay of labeled receptors during the chase was linear on a semilog plot, consistent with first order kinetics. Receptor half-life was 9 h when cells were cultured in either phenol red-containing medium supplemented with fetal calf serum or in phenol red free-medium supplemented with charcoal extracted serum, indicating that endogenous steroids do not affect receptor half-life. Receptor half-life was also unchanged when cells were cultured in the presence of 0.1 microM dexamethasone, a glucocorticoid hormone, or 0.1 microM RU486 (11 beta-(4-dimethylamino-phenyl)-17 beta-hydroxy-17 alpha-(propynylestra-4,9- diene-3-one), an antiglucocorticoid hormone. We conclude that the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells is not regulated by either glucocorticoid or antiglucocorticoid hormones.     

Rapid turnover of low-density lipoprotein receptor by a non-lysosomal pathway in mouse macrophage J774 cells and inhibitory effect of brefeldin A

The low-density lipoprotein (LDL) receptor of molecular mass 155 kDa was expressed on the cell surface of cultured mouse macrophage J774 cells. The conversion rate of precursor to mature form of LDL receptor in J774 cells was comparable to that in mouse fibroblast L cells. The half-life of the LDL receptor of J774 cells was about 2 h, that of L cells was about 11 h. The rapid degradation of LDL receptor was not significantly inhibited by the lysosomotropic agents, chloroquine and NH4Cl, nor by the thiol-protease inhibitors leupeptin and E-64. By contrast, incubation at 18 degrees C retarded the degradation of LDL receptor. Treatment of J774 cells with brefeldin A, an inhibitor of membrane transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the rapid turnover of the LDL receptor. Even after a 9-h chase in the presence of brefeldin A, LDL receptor 5-10 kDa smaller than the normal mature form was found to be stable. Rapid turnover of the LDL receptor in the macrophages appeared to occur after exit from the Golgi apparatus, possibly during transport of the LDL receptor to the plasma membrane.     

The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes.

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.     

Stability of cytoplasmic ribonucleic acid in a mouse myeloma: estimation of the half-life of the messenger RNA coding for an immunoglobulin light chain

This paper describes experiments in which the half-lives of a number of cytoplasmic RNA species have been estimated in a mouse myeloma (MOPC 21) without resort to metabolic inhibitors. Partial purification of the messenger RNA coding for immunoglobulin light chains enabled an estimate of the stability of this species to be made. The procedure chosen was that of a conventional pulse-chase following uniform labelling of cells with [³H]uridine. Centrifugation of the uniformly labelled cells and resuspension in 0·1 mm-uridine resulted in a 75% drop in the specific activity of the UTP pool within 2 hours, followed by a logarithmic decay with a half-life of about 3·5 hours. Exposure of P3K cells to uridine causes them to swell appreciably and centrifugation at the end of the pulse period is followed by a lag phase of 3 hours before the cells re-enter logarithmic growth. Since all chase conditions had certain disadvantages, a comparison of experiments using different chase conditions was undertaken. The stability of the various RNA species did not vary greatly under the different chase conditions. The half-life of the light-chain mRNA is estimated to be 12 to 14 hours, although a value in the range of 5 to 20 hours cannot be excluded. An RNA fraction including the heavy-chain mRNA behaves similarly. Half-lives determined for other RNA species were: 18 S ribosomal RNA (40 to 60 h); 12 S mitochondrial ribosomal RNA (28 to 32 h). Poly(A)-containing RNA from free polyribosomes decays rapidly in the first 5 hours with a half-life of 20 to 30 hours, subsequently.     

Rapid degradation of steroid sulfatase in multiple sulfatase deficiency

Pulse labeling followed by SDS-PAGE electrophoresis of immunoprecipitated [35S]methionine-labeled steroid sulfatase (STS) gave a single band of molecular weight 65,000 daltons. After a chase period of 18 hours the material appeared as molecular weight approximately 64,000. No labeled STS could be detected in fibroblasts from individuals with STS deficient X-linked ichthyosis. Pulse-chase labeling of normal and multiple sulfatase deficiency (MSD) fibroblasts showed a normal rate of synthesis of STS in MSD during a 3 hour pulse but during the chase the STS of MSD cells disappeared with a half-life of 4 to 6 hours until approximately 25% of the material remained after 24 hr. STS of normal cells had a half-life of 6 days. The material produced in MSD cells had the same molecular size as normal and had the same amount of endoglycosidase sensitive carbohydrate as normal. The defect in MSD thus seems to result in degradation after the addition of N-linked oligosaccharides.     

Kinetic behavior of 30S rRNA intermediate labeled in developing embryonic cells of Xenopus laevis

In Xenopus neurula cells, "30S" RNA was found to be labeled with 3H-uridine after a relatively short labeling period. Results obtained from cumulative labeling and pulse-labeling and chase experiments with cells from late gastrulae, yolk plug-stage embryos, and neurulae showed that the 30S RNA is an intermediate in rRNA processing and is derived from 40S pre-rRNA and processed to 28S rRNA. The half-life of the 30S rRNA intermediate was about 7.5 min or less at the three stages examined.     

Biosynthesis and stability of globin mRNA in cultured erythroleukemic friend cells

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with ³H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA covalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2–4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16–17 hr. The rest of the poly(A)-containing RNA was composed of two kinetic populations: 85–90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but if subsequently declined gradually.Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast.

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.     

The effect of monensin on thyroglobulin secretion

The effect of monensin on the secretion of thyroglobulin was studied in open follicles isolated from pig thyroid tissue; in this system, thyroglobulin is secreted into the incubation medium. When monensin was present during a 4-h chase incubation after pulse-labelling with 3H-leucine, the secretion of labelled thyroglobulin was reduced by about 85%; in electron-microscopic autoradiographs of rat thyroid lobes labelled and chase-incubated under similar conditions the relative number of grains over follicle lumina was strongly reduced when monensin was present during the chase. These observations are in agreement with the consensus that monensin arrests transport of secretory proteins in the Golgi complex. In other experiments, pulse-labelled follicles were chase-incubated for 1.5 h whereby labelled thyroglobulin was transported from the RER to exocytic vesicles. Monensin present during a subsequent chase of 0.5 h caused only a moderate decrease of labelled thyroglobulin secretion. TSH present during the second chase-stimulated secretion in both control and monensin-exposed follicles. TSH also caused a drastic reduction of exocytic vesicles in rat thyroid lobes, and the number of vesicles remaining in the cells was the same in controls and lobes exposed to the ionophore. The observations are interpreted to show that monensin does not inhibit the basal or TSH-stimulated transport of thyroglobulin from the site of monensin-induced arrest in the Golgi complex to the apical cell surface or the exocytosis of thyroglobulin.     

Selective Degradation of Newly Synthesized Nonmessenger Simian Virus 40 Transcripts

By pretreating simian virus 40-infected BSC-1 cells with glucosamine, [(3)H]uridine labeling of both cellular and viral RNA can be halted instantaneously by addition of cold uridine. We have studied the fate of pulse-labeled viral RNA from cells at 45 h postinfection under these conditions. During a 5-min period of labeling, both the messenger and nonmessenger regions of the late strand were transcribed. After various chase periods, nuclear viral species which sediment at 19, 17.5, and 16S were observed. Nuclear viral RNA decays in a multiphasic manner. Of the material present at the beginning of the chase period, 50% was degraded rapidly with a half-life of 8 min (initial processing). This rapidly degraded material was that fraction of the late strand which did not give rise to stable late mRNA species. Forty percent was transported to the cytoplasm, and 10% remained in the nucleus as material which sedimented in the 2 to 4S region. These 2 to 4S viral RNAs had a half-life of 3 h, and hybridization studies suggest that they are in part coded for by the late-strand nonmessenger region and are derived from the initial nuclear processing step. Another part is coded for by the late-strand messenger region and may be generated by some subsequent nuclear cleavages of 19S RNA into 17.5 and 16S RNAs. Transport of nuclear viral RNA into the cytoplasm was detected after a 5-min pulse and a 7-min chase. The maximum amount of labeled viral RNA was accumulated in the cytoplasm after a 30-min to 1-h chase. At least two viral cytoplasmic species were observed. Kinetic data suggest that 19S RNA is transported directly from the nucleus. Whether cytoplasmic 16S is formed by cleavage of 19S RNA in the cytoplasm is not clear. The half-lives of cytoplasmic 19 and 16S RNAs can be approximated as 2 and 5 h, respectively.     

Metabolism of polyadenylated mRNA in growing human lymphocytes.

The kinetics of degradation of newly synthesized, cytoplasmic polyadenylated RNA have been examined in normal human lymphocytes stimulated to grow with phytohemagglutinin. A single class of poly(A)-bearing RNA was identified with a half-life of approximately 50 h. In the presence of actinomycin D, the half-life was 5 to 6 h, and virtually no decay of pulse-labeled material was detectable after 6 h of chase incubation with cordycepin. These findings contrast sharply with data obtained from other growing human cells used as controls: polyadenylated mRNA in MOLT-4 cells, a cultured line of T lymphocytes, had a half-life of 2 h in the presence of actinomycin D. The stability of poly(A)-containing RNA in stimulated lymphocytes from normal donors is therefore not simply a manifestation of cell proliferation. In normal resting lymphocytes, Berger and Copper [(1975) Proc. Natl. Acad. Sci. U.S. 72, 3873--3877] reported the existence of 2 classes of polyadenylated mRNA with half-lives of under an hour and greater than 20 h, respectively. Since short-lived poly(A)-bearing mRNA is absent from mitogen-stimulated lymphocytes, the data suggest that stabilization of previously labile poly(A)-bearing RNA is one of many carefully regulated processes accompanying growth induction in normal lymphoid cells.     

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

Matrix association of early- and late-replicating chromatin studied by single-cell electrophoresis

CHO-K1 cells were synchronized at the G(1)/S border by mitotic shake-off and aphidicolin incubation. Pulse-labeling with tritium was done at 30 min, 2 or 5 h into the S-phase, with chase incubations for different times in non-radioactive medium. The cells were subjected to neutral microelectrophoresis to extend the DNA into "comets," after which the label was visualized through autoradiography. At zero chase time, all label was positioned in the head. The displacement of label into the tails increased with time, reaching a maximum at about 5 h after the pulse. A lag phase of 2-3 h was observed for the early-labeled cells before the displacement started. Also, more label was released after overnight serum starvation, but this was reversed through a 3-h incubation at normal growth conditions. It was found that late-replicating chromatin is organized in larger domains than early-replicating chromatin, and DNA polymerase seems to be an important organizer. Early-replicating chromatin has other important attachments to the nuclear matrix, dependent on metabolic activity.     

Cell-surface glycosaminoglycans: Turnover in cultured human embryo fibroblasts (IMR-90)

As constituents of both extracellular matrix and the cell surface, glycosaminoglycans are in a strategic position to influence several basic cell features. The localization and turnover of glycosaminoglycans was investigated in cultured normal human embryo fibroblasts of lung origin (IMR-90). Attention was directed particularly toward that compartment of the culture which could be released by gentle proteloysis (trypsin, 0.1 mg/ml, 15 min) and is considered to represent the cell surface. In the presence of Na₂SO₄, sulfated glycosaminoglycans (S-GAGs) of the cell surface were labeled rapidly, but within 30 min some ³⁵S-GAG appeared in the extracellular medium. The intracellular pool of S-GAGs labeled during a 10-min period was lost during the first hr of chase with a half-life of 18 min, compared with 16 hr for S-GAGs labeled over a 48-hr period. Pulse-labeled S-GAGs of the surface turned over with an initial half-life of 60 min, compared with 7 hr for surface material labeled over a 48-hr period. These rapid movements of the early chase period were followed by similar movement at a much slower rate. The results are consistent with a model in which most of the S-GAGs synthesized in the cell move rapidly to the surface. The surface GAGs are then released immediately to the medium or accumulate at the membrane to be shed more slowly at a later time or to be degraded. The S-GAG which left the cell layer most rapidly during chase was dermatan sulfate, while heparan sulfate made up an increasing percentage of the cell layer as chase progressed. These cultures produce a fibrillar matrix of fibronectin, but the kinetics of this study suggest that the S-GAGs of the surface are membrane-bound, and an extracellular glycosaminoglycan matrix does not form.     

Accelerated secretion of human lysozyme with a disulfide bond mutation.

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.