Synthesis Of Secretory Proteins Research Articles

Age-related changes in the cellular level of amylase and protein synthesis in the rat parotid gland.

Age-related changes in the cellular content of secretory proteins and protein synthesis were studied in parotid glands of rats of various ages. The secretory protein content (determined by measuring the level of alpha-amylase activity) and the synthesis of proteins (assayed by the rate of incorporation of 3H-leucine into acid-insoluble proteins) decline with increasing age. Morphological and radioautographic studies of the gland indicate that the decline in protein synthesis is due to the reduction in the ability of secretory cells to synthesize proteins.

Developmental regulation of secretory protein synthesis in rat seminal vesicle.

This paper reports a developmental study of one of the major secretory proteins (SVS IV) of the rat seminal vesicle. Measured by radioimmunoassay, SVS IV is detectable even in very young rats (5 days old) but does not begin to accumulate in massive amounts until animals reach about 35 days of age. We have compared SVS IV synthesis by minced tissue from rats undergoing sexual maturation (25-60 days of age) with levels of translatable SVS IV mRNA present. Between 25 and 60 days of age, the proportion of newly synthesized protein devoted to SVS IV by tissue minces increased from 0.8% to 20%. In contrast, the proportion of total RNA devoted to translatable SVS IV mRNA was already high in the immature glands and increased less than 4-fold during development. These results seem to indicate that factors other than the presence of potentially translatable message regulate the rate of net synthesis of SVS IV during seminal vesicle maturation.

Effect of fibrinogenolytic products D and E on fibrinogen and albumin synthesis in the rat.

Previous studies are in conflict over the effect of infusing mixed fibrinogen-fibrin degradation products on fibrinogen synthesis, as determined by changes in fibrinogen concentration or by incorporation of labeled amino acids into fibrinogen. We have injected purified homologous fragments D1 and E into rats and measured their fibrinogen and albumin synthetic rates by the [14C]carbonate technique, a method that provides quantitative estimates of hepatic secretory protein synthesis. Fibrinogen fractional synthetic rates were increased 2.5 times in animals injected with fragment D1, compared with saline-injected controls. No increase were observed in fragment E-injected animals. Neither fragment produced changes in albumin synthesis. Fragment D increased plasma fibrinogen concentration, but did not raise plasma haptoglobin levels. These results suggest that fragment D is a regulator of fibrinogen synthesis.

Quantitative Analysis of Protein Synthesis Altered by Estrogen in Cultured Xenopus Liver Parenchymal Cell.

Using the primary culture system of male Xenopus laevis hepatocytes consisting of more than 95% parenchymal cells, the effect of estradiol-17 β (10-6 M) on protein synthesis was quantitatively analyzed by 3 H-leucine incorporation kinetics and the estimation of specific radioactivity of newly synthesized secretory protein. The cells in a well defined culture revealed high plating efficiency and very low DNA synthetic activity. The cultured cells could synthesize several secretory proteins containing serum albumin. The pattern of secreted proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) did not alter with culture time but secretion rate of protein increased for 7 days, starting on the third day following inoculation. Estradiol added to the culture media extensively induced the synthesis of yolk precursor protein vitellogenin which accounted for 40-50% of the overall secretory protein synthesis and 20-30% of the total protein synthesis on day 7 of estradiol treatment. Ultimately, the total protein synthesis and secretory protein synthesis were stimulated 1.2-1.3 fold and 2.0-2.2 fold, respectively, over those of the control cells cultured in the absence of estradiol. These results indicated that the stimulation of protein synthesis was largely due to vitellogenin production. Such an estradiol-dependent stimulation of protein synthesis was also detected in the low molecular weight protein(s). On the other hand, albumin synthesis was evidently reduced by estradiol. Thus, estradiol had two different effects on protein synthesis. The results obtained in this study will be discussed in relation to the findings o in vivo experiments.

Protein synthesis during salivary gland development, ageing, and cell death in Chironomus tentans larvae

Salivary gland protein synthesis in Chironomus tentans larvae was analyzed from the mid-third instar to larval pupation. Correcting for stage specific variations in the specific activity of the amino acid pool revealed a 30–40% reduction in the rate of protein synthesis during the larval moult. Except for a transient increase early in the fourth instar, this low rate of protein synthesis was maintained until the pharate pupal period when protein synthesis dramatically increased: maximum synthesis occurred in mid-pharate pupae with a subsequent decline correlating with gland autolysis and cell death at pupation. Each developmental period was characterized by a particular pattern of secretory protein synthesis: high and 35,000 daltons peptides were maximally synthesized only at particular larval stages, being reduced or absent in post-ecdysis, diapause and autolysing salivary glands. Except for the ecdysone puffs, and as otherwise previously noted ( Clever, 1961, 1962), puffing activity during the peri-moult period remained relatively constant and did not decrease by the 30–40% predicted from the decreased rate of protein synthesis. The nearly complete loss in synthesis of the 35,000 daltons peptide was not accompanied by regression in any puff.

Intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus

Rat hepatoma cells which secrete several serum proteins have been infected with a temperature sensitive mutant of Vesicular Stomatitis Virus, which is defective in RNA replication and transcription. These infected cells, grown at 39.5°C, continue normal secretion of albumin and transferrin, so that synthesis and maturation of secretory and membrane proteins, including the VSV G protein, can be studied in the same cells. Serum proteins are not secreted from the cells at the same rate; half of the newly synthesized albumin appears in the medium after 23 minutes, while half of the glycoprotein transferrin is secreted only after about 40 minutes. By contrast, two membrane proteins—the VSV G glycoprotein and a high molecular weight hepatoma surface protein—are detected at the cell surface approximately 23 minutes after their synthesis. The differences in the rate of externalization among the secretory and membrane glycoproteins are due to differences in the rate of transport from the rough endoplasmic reticulum (ER) into or through the Golgi region, as judged by the time required for maturation of the asn-linked oligosaccharides. Unglycosylated VSV G does not reach the cell surface, while unglycosylated transferrin is secreted at the same rate as the glycosylated species. Thus neither the synthesis nor the presence of the asn-linked oligosaccharide chain are determinants in the rate of transferrin secretion. The monovalent ionophore monensin inhibits both secretion of serum proteins and migration of membrane proteins to the cell surface. However, the intracellular transport of transferrin is blocked in a stage where the molecule is still sensitive to endo-β-N-acetylglucosaminidase H, while VSV G reaches a stage in which it is resistant to the enzyme. These results indicate that at least part of the intracellular pathway may be different for membrane and secretory proteins and glycoproteins.

THE SECRETORY CYCLE OF DIONAEA MUSCIPULA ELLIS.

SummaryThe cellular mechanisms involved in the storage and synthesis of secretory proteins in the glands of the carnivorous plant Dionaea muscipula were examined. Important roles for a sub‐compartment of the vacuole and smooth endoplasmic reticulum were demonstrated by high‐resolution autoradiography and cytochemistry. It is shown, for the first time in a carnivorous plant, that de novo protein synthesis occurs during the secretory phase of activity and that part of this protein is directly secreted.

Alcohol and very low density lipoprotein synthesis and secretion by isolated hepatocytes.

Labeled leucine can be used to measure accurately the rate of both total and secretory protein synthesis by isolated hepatocytes if at least 1 mM leucine is added to the incubation medium, even in the presence of 50 mM ethanol. Using this technique it was found that ethanol caused a significant inhibition of very low density lipoprotein (VLDL) as well as total protein synthetic rates in hepatocytes from both fed and fasted rats. In contrast, a single acute oral dose of ethanol to fasted rats caused within 4 hr a threefold stimulation in the rate of VLDL synthesis without affecting the total protein synthetic rate in the hepatocyte system.

Proteins synthesized and secreted during rat pancreatic development.

The synthesis and secretion of proteins during development of the pancreas was analyzed using two-dimensional gel electrophoresis. The pattern of synthesis of the total proteins of the pancreas was found to change very little from 14 to 18 d gestation. In addition, the protein synthetic pattern of the embryonic pancreas was very similar to the protein patterns of several other embryonic tissues (gut, lung, and mesenchyme). Between 18 d gestation and the adult stage, the synthesis of the majority of protein species fades as the synthesis of the secretory (pro)enzymes becomes dominant. Thus, the terminal differentiation of the pancreas appears to involve the dominant expression of a limited set of genes (coding, in part, for the digestive [pro]enzymes) while the pattern of expression of the remaining domain remains relatively unchanged. Many of the secretory (pro)enzymes were identified and their synthesis during development was monitored. The synthesis of several secretory proteins was detected between 15 and 18 d gestation (e.g., amylase and chymotrypsinogen), whereas the synthesis of others was not detected until after 18 d gestation (i.e., trypsinogen, ribonuclease, proelastase, and lipase). Between 18 d gestation and the adult stage, the synthesis of the digestive (pro)enzymes increases to > 90% of pancreatic protein synthesis. The secretion of digestive (pro)enzymes was detected as early as 15 d gestation. The selective release of a second set of proteins was detected in the early embryo. These proteins are not detected in the adult pancreas or in zymogen granules but are also released by several other embryonic tissues. The function of this set of proteins is unknown.

Preferential response of thyroid glycosyltransferases to changes in thyrotropin stimulation

The level of thyrotropin stimulation of rat thyroid was modified to permit a study of the regulation of some of the enzymes of this tissue. This was accomplished by the administration of either propylthiouracil to increase the endogenous thyrotropin levels or thyroxine to suppress production of the trophic hormone. The enzymes measured included two glycosyltransferases involved in the synthesis of the main secretory protein of the gland (thyroglobulin), two lysosomal enzymes which may contribute to its catabolism, as well as two other enzymes of a more general nature. In the propylthiouracil-treated animals changes in the activities of succinic dehydrogenase, protease, and 5′-nucleotidase corresponded to the increase in the weight of the gland and appeared to be nonspecific in nature; these three enzymes, moreover, showed no changes after thyroxine treatment. The level of the N-acetylglucosaminidase was significantly decreased, per gram of tissue, in both groups of treated animals. The only enzymes which appeared to be specifically affected by the modulation of thyrotropin stimulation were the glycosyltransferases, with both mannosyl- and galactosyltransferases showing an increase in the thyroids of the propylthiouracil-treated animals and a decrease in those treated with thyroxine. This suggests that post-translational steps, such as carbohydration, may play an important role in regulating the turnover of thyroglobulin and therefore influence the overall rate of thyroid hormone formation. The distribution of each of the enzymes between the soluble and particulate fractions of the tissue was also measured and it was noted that the glycosyltransferases, which showed the most marked increase in total activity as a result of thyrotropin stimulation, also showed a statistically significant increase in the percentage present in the particle-bound form.

The inhibitory effects of sodium salicylate on synthesis of factor VII by the perfused rat liver

The isolated rat liver perfusion system has been used to study the effects of sodium salicylate on the synthesis of a vitamin K-dependent coagulation factor (Factor VII) as well as other liver secretory proteins which are not felt to be vitamin K-dependent: fibrinogen, albumin and antithrombin III. Assuming a definition for units of Factor VII activity (measured with a Factor VII deficient substrate) as 100 units per ml normal rat plasma, control liver perfusions of 10 hours duration showed net cumulative synthesis values of 1580 ± 320 units Factor VII activity/300 cm 2 body surface area of the rat liver donor. The addition of different concentrations of sodium salicylate (15 mg or 25 mg) produced a dose related inhibition of Factor VII synthesis. At the higher concentration of sodium salicylate (25 mg) inhibition of Factor VII synthesis was comparable to that observed in perfusions where protein synthesis was completely blocked with puromycin. The synthesis of other liver secretory proteins, albumin, fibrinogen and antithrombin III was not altered by the presence of salicylate. The effect of sodium salicylate on Factor VII synthesis was apparent even when rat liver donors were pretreated with vitamin K and supplemental vitamin K was added directly to the perfusate.

Dexamethasone regulates the program of secretory glycoprotein synthesis in hepatoma tissue culture cells.

The secretory glycoproteins synthesized by hepatoma tissue culture (HTC) cells were resolved by two-dimensional polyacrylamide gel electrophoresis of media from cells that were grown in the presence of [(3)H]fucose. These cells synthesize and secrete a complex set of fucose-containing glycoproteins. These secretory glycoproteins are distinct from those glycoproteins present in the plasma membrane of HTC cells. Incubation of HTC cells with dexamethasone has a pronounced effect on the quality and quantity (denoted here as the program) of secretory protein synthesis, as assayed by the short-term incorporation of labeled mannose, fucose, or methionine. The synthesis of two mannose- and fucose- containing glycoprotein series, one of 50,000 mol wt and a more heterogeneous series with mol wt of 35,000-50,000, is increased to a high level by the hormone; conversely, the synthesis of other secretory proteins, particularly one with mol wt of 70,000, is decreased or stopped completely. The synthesis of some major secretory proteins is not affected by the hormone. Dexamethasone has less of an effect on the composition of either total cell membrane glycoprotein or plasma membrane glycoprotein. But there is a decrease in the synthesis of a major membrane glycoprotein series with mol wt of 140,000. These effects of dexamethasone are relatively specific to HTC cells. Neither Reuber H-35 cells nor primary cultures of rat hepatocytes show the same response to the steroid. Two variant HTC cell lines, which were selected for their resistance to dexamethasone inhibition of extracellular plasminogen activator activity, respond only partially to the steroid-induced regulation of the secretory and membrane glycoproteins.

Effect of fasting and feeding on synthesis and intracellular transport of proteins in the frog exocrine pancreas.

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.

Correlation of albumin production rates and albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic rats.

We have studied the effects of alloxan-induced diabetes and subsequent insulin replacement on albumin and total hepatic protein synthesis. Diabetes resulted in a reduction to approximately 20% of normal in albumin synthesis relative to the rate of total protein synthesis in vivo and a reduction to 10% in the absolute rate of albumin secretion by perfused livers. In contrast, the synthesis of total secretory protein and retained hepatic protein was affected to a lesser extent by diabetes. Treatment of diabetic rats with insulin restored rates of albumin and total hepatic protein synthesis to normal levels. The molecular basis of these alterations in albumin synthesis was investigated by examining albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic animals. The level of albumin mRNA, whether assayed by cell-free translation or by hybridization to a specific complementary DNA probe, was markedly decreased in livers of diabetic animals and was restored to normal by insulin treatment. These changes occurred in parallel with changes in the rates of albumin secretion observed in perfused liver, suggesting that albumin mRNA content is the primary factor responsible for altering rates of albumin synthesis under these conditions.

The effects of calcium and magnesium on the secretion of parathormone and parathyroid secretory protein by isolated porcine parathyroid cells.

The preparation of dispersed parathyroid cells by collagenase digestion of porcine parathyroid glands, essentially as outlined by Brown et al. (Endocrinology 99: 1582, 1976), is described. The cells secrete parathormone linearly for at least 4 h of incubation and rapidly respond in inverse fashion to changes in the medium calcium and magnesium concentrations over the range 0.5-3.0 mM. In terms of inhibition of secretion, either ion was more effective in the presence of a minimum concentration of the other, indicating that calcium and magnesium affect separate cellular sites. Parathormone was identified both by immunoassay of the whole incubation medium and by its separation by polyacrylamide gels and carboxymethylcellulose chromatography. When the cells were incubated with radioactive amino acids and both the medium and cells were subsequently analyzed on gels, we found that parathyroid secretory protein as well as parathormone and some immunoactive fragments were present. Analysis of the radioactive protein contained in the cells at high and low calcium concentrations revealed that calcium decreased the formation of the secretory protein by approximately 40% without appreciably affecting the formation of proparathormone or parathormone. The secretion of both parathyroid secretory protein and parathormone were inversely proportional to the concentrations of medium calcium or magnesium. The secretion of the latter, however, was more sensitive (95% inhibition) than parathormone (40-60% inhibition) to changes in medium divalent cations. These results suggest that the synthesis, intracellular processing, or secretion of parathormone and parathyroid secretory protein utilize independent calcium- and magnesium-regulated pathways.

Asynchrony in the synthesis of secretory proteins in the venom gland of the snake Vipera palaestinae.

1. Venom of Vipera palastinae was subjected to isoelectrofocusing on polyacrylamide gel. The protein separation profiles were similar for different venom samples; more than 25 protein bands with a wide range of pI values could be demonstrated by this technique. 2. Labelled venom was obtained 8h after an intracardial injection of [3H]leucine. The relative radioactivities of four out of 12 main protein bands were significantly different in the venom synthesized during the 2nd day of the venom regeneration cycle as compared with the venom of the 4th day. The comparison was made in venom samples obtained from the two glands of the same snake at two different secretory stages. 3. It is concluded that the asynchronous synthesis of exportable proteins after the initiation of a new venom regeneration cycle is responsible for the non-parallel secretion of some venom proteins by the venom gland of Vipera palaestinae during the first few days after milking.

Protein transfer across microsomal membranes reassembled from separated membrane components.

THE synthesis of secretory proteins is initiated on ribosomes in the cell cytoplasm and the first part of the polypeptide chain to appear is a short sequence termed the signal sequence1. This signal is thought to direct the ribosomal complex to the endoplasmic reticulum (ER) membrane where the synthesis of the rest of the secretory protein is tightly coupled to its transfer across the membrane into the cisternal space. In all but one case, it is clear that the polypeptide chain is processed during transfer to remove the signal sequence2,3. Little is known about the membrane proteins involved in conveying the growing polypeptide chain across the membrane. A classical approach to learning more about these proteins would be to dissect the microsomal membrane into inactive components that can be reassembled subsequently into a functional entity. This would allow the purification of components involved in protein transfer. Here we report the first such reassembly.

Membranes of mammary gland—XVIII. 2-deoxy- d-glucose and 5-thio- d-glucose decrease lactose content, inhibit secretory maturation and depress protein synthesis and secretion in lactating rat mammary gland

1. 1. Oral administration of either 2-deoxy- d-glucose or 5-thio- d-glucose to lactating rats decreased mammary tissue lactose content to half of control levels within 3 hr. 2. 2. While secretory vesicles in control rats were large and fully swollen in apical regions of epithelial cells, those in epithelial cells of glucose-analog treated rats were less fully developed. 3. 3. Mammary tissue from analog treated rats synthesized protein at lower rates than that from control animals. 4. 4. When included in the incubation medium deoxyglucose and thioglucose blocked release of protein. 5. 5. The effects of these glucose analogs on lactose synthesis, secretory vesicle development and protein synthesis and release appeared to be related to their ability to inhibit glucose transport.

Plasma protein synthesis by isolated rat hepatocytes.

A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.