Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755

Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. The phytase (phyC) from Bacillus subtilis VTT E-68013 was expressed in Lactobacillus plantarum strain 755 using Lact. amylovorus alpha-amylase secretion signals. In an overnight cultivation in MRS medium containing cellobiose for induction of the alpha-amylase promoter, catalytically active phytase was secreted as a predominant extracellular protein. However, Western blot analysis revealed unprocessed and processed phytase in the cell fraction. Pulse chase experiments showed that the recombinant phytase was secreted at a slower rate in comparison to the native proteins of Lact. plantarum 755.     

Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes

Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorporation of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBH with an intra-cellular retention half-time of 50 min (initial half-life of decay, t1/2 = 25 min) compared with 85 min in control (t1/2 = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin-treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively. When hepatocyte monolayers were continuously labeled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBH was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase. Insulin inhibited the secretion of immunoassayable apoB but not labeled apoB. Results demonstrate that 1) insulin inhibits synthesis of apoB from [35S]methionine, 2) insulin stimulates degradation of freshly translated apoB favoring apoBH over apoBL, and 3) an intracellular pool of apoB, primarily apoBL, exists that is largely unaffected by insulin. Overall, insulin action in primary hepatocyte cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.     

Batch production and secretion kinetics of hybridomas: Pulse-chase experiments

Pulse chase experiments of two mouse hybridoma lines were conducted in order to elucidate the kinetics of monoclonal antibody (mAb) production and secretion during different stages of batch cultures. The results indicate that a stock of cytoplasmic IgG exists in hybridoma cells and that the concentration of this stored IgG depends on the cell line used and the stage of the culture. This stored IgG can be released by dying cells, and a certain quantity of the secreted IgG is derived from this source. However, only between 0.3 and 9.3% of the released IgG of U0208 (average: 2.08%) and between 2.08 and 25.8% of the IgG, released from I.13.17 (average: 6.95%), were of storage origin, calculated on culture viability and intracellular IgG-stock. Comparing the accumulation of radio-labelled IgG (IgG^*) in the supernatant with the reduction of cytoplasmic IgG^* during the chase experiments, the percentages range between 14 and 50%, somewhat higher values probably caused by changes in the culture conditions. These changes led to a release of IgG during the chase experiments, which accounts for about 20–25% of the totally secreted IgG.It could be established that during the logarithmic growth phase of batch cultures a certain percentage of synthesized IgG was not released but stored within the cells: for U0208: 0.3–4.5%, for I.13.17: 1–7.6%. During the stationary and death phase, this percentage ranged between 1.5 and 20% for U0208 and between 0.5 and 8.1% for I.13.17. Finally, the chase experiments also revealed that the time of synthesis, assembly, and secretion of mAbs does not vary much during the different phases of batch cultures, and is within the range of 1.5 and 3 hrs.     

Effects of 5'deoxy-5'-methylthioadenosine on the metabolism of S-adenosyl methionine

The treatment of transformed rat cells with micromolar amounts of 5'deoxy 5'methyl thioadenosine induces rapid effects on the rate of methylation of DNA concomitantly with alterations of intracellular pools of S-adenosyl methionine and S-adenosyl homocysteine. Pulse chase labelling experiments indicate that 5'deoxy 5'methylthioadenosine does not inhibit the degradation of S-adenosyl homocysteine but inhibits the consumption of S-adenosyl methionine. In vitro transmethylation assays performed with heterologous DNA show that low doses of the thioethernucleoside do not significantly affect the DNA methyltransferase activity of cellular extracts. The biological role of 5'deoxy 5'methylthioadenosine, a natural molecule formed during the synthesis of polyamines is discussed.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

hFE65L Influences Amyloid Precursor Protein Maturation and Secretion

The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective alpha-secretase-derived secreted APP (APPs alpha) and the Alzheimer's disease-associated beta-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a three-fold increase in the amount of APPs alpha recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs alpha secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.     

Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes.

The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.     

Biosynthesis of chick type VI collagen. II. Processing and secretion in fibroblasts and smooth muscle cells

The biosynthesis of type VI collagen was studied in "matrix-free" chick embryo smooth muscle cells and fibroblasts. Omission of ascorbate from the culture affected to a great extent the secretion in fibroblasts but had a very minor effect on smooth muscle cells. Quantitative analysis of the secretion process in continuous time course and in pulse-chase experiments confirmed that fibroblasts and smooth muscle cells secreted type VI collagen with the same chain composition but with different kinetics: after 4 h of chase more than 60% of the labeled type VI collagen was present in the culture medium of fibroblasts, whereas at the same time interval less than 25% was secreted by smooth muscle cells. The different kinetics depends on intrinsic properties of the cells, since it was detected also in adherent cells. However, even in fibroblasts, secretion of type VI collagen was much slower than secretion of fibronectin, of which more than 50% was already in the cell medium after 1 h of chase. Treatment of the cells with inhibitors of hydroxylation and glycosylation caused a shift in mobility that revealed a size heterogeneity in the Mr = 260,000 subunit. No evidence of processing was observed in chick cells for any of the subunits that were synthesized and secreted uncleaved. In addition, after several days of chase the Mr of the subunits of type VI collagen isolated from the matrix remained unchanged, thus excluding that in the chick even a partial or incomplete processing takes place.     

Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.     

Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells

The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains.     

Monitoring transport of a recombinant phosphotriesterase in Streptomyces lividans using a pulse-chase system

A heterologous phosphotriesterase (parathion hydrolase) was previosly shown to be secreted by Streptomyces lividans. To investigate the mechanism of secretion, a system to label the protein and follow its expression and secretion was developed. The recombinant S. lividans was grown first in a defined medium containing [³⁵S]methionine that permitted expression but not secretion. It was then transferred to tryptone/glucose medium with unlabeled methionine for the chase period, during which secretion was observed. The results indicate a relatively slow rate of secretion that is also dependent on the growth medium.     

Heparin decreases the degradation rate of lipoprotein lipase in adipocytes

The mechanism responsible for the stimulation of secretion of lipoprotein lipase by heparin in cultured cells was studied with avian adipocytes in culture. Immunoprecipitation followed by electrophoresis and fluorography were used to isolate and quantitate the radiolabeled enzyme, whereas total lipoprotein lipase was quantitated by radioimmunoassay. Rates of synthesis of lipoprotein lipase were not different for control or heparin treatments as judged by incorporation of L-[35S]methionine counts into lipoprotein lipase during a 20-min pulse. This observation was corroborated in pulse-chase experiments where the calculation of total lipoprotein lipase synthesis, based on the rate of change in enzyme-specific activity during the chase, showed no difference between control (8.13 +/- 3.1) and heparin treatments (9.1 +/- 5.3 ng/h/60-mm dish). Secretion rates of enzyme were calculated from measurements of the radioactivity of the secreted enzyme and the cellular enzyme-specific activity. Degradation rates were calculated by difference between synthesis and secretion rates of enzyme. In control cells 76% of the synthesized enzyme was degraded. Addition of heparin to the culture medium reduced the degradation rate to 21% of the synthetic rate. The presence of heparin in cell media resulted in a decrease in apparent intracellular retention half-time for secreted enzyme from 160 +/- 44 min to 25 +/- 1 min. The above data demonstrate that the increase in lipoprotein lipase protein secretion, observed upon addition of heparin to cultured adipocytes, is due to a decreased degradation rate with no change in synthetic rate. Finally, newly synthesized lipoprotein lipase in cultured adipocytes is secreted constitutively and there is no evidence that it is stored in an intracellular pool.     

Differential trafficking of soluble and integral membrane secretory granule-associated proteins

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.     

Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media were analysed using N-terminal sequence analysis. In a submerged wheat-based growth medium of A. oryzae, besides alpha-amylase, also an arabinosidase and xylanase were abundantly produced. In the extracts of A. oryzae grown on wheat-based solid substrate besides alpha-amylase and chitinase, two new proteins of 16 and 27 kDa were identified. These hypothetical proteins showed only close homologies to filamentous fungal proteins.     

Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding.

Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H-sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.     

A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.     

Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.     

Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.