Amylase Synthesis Research Articles

Gibberellic Acid-Induced Alpha -Amylase Synthesis in Wheat Aleurone Layers: a Distinction Between Site Saturation and Lag Times.

We have examined a number of parameters relating to the gibberellic acid (GA3) requirement for the induction process which leads to the formation of �-amylase in wheat aleurone tissue. (1) Over a wide range of hormone concentrations, aleurone tissue was found to produce less �-amylase after hormone withdrawal by washing at a time soon after GA*3 application. (2) The approximate length of this washing-sensitive period was found to vary inversely to the applied GA*3 concentration. (3) Following this early period, a time was found when the response could not be lowered by this same washing procedure which had earlier lowered the response. (4) Separate experiments determined that these results could not be accounted for by variation in the lag/latent period, since the lag period was about the same for a wide range of GA*3 concentrations. To help explain these results we have suggested the term 'site saturation time' which is separate from but intimately involved with the lagllatent period. This term is defined as the time required for the saturation and activation of all sites which ultimately lead to the production of �-amylase. It is suggested that site saturation time must be taken into account when the early events of �-amylase induction are examined.

Effects of heavy metals on enzyme synthesis in substrate-amended river sediments

The effects of heavy metals in diverse substrateamended river sediments were studied. Cd2+, Pb2+ and Zn2+ generally had a marked inhibitory effect on the synthesis of amylase, cellulase and urease, on numbers of substrate-hydrolysing bacteria, in all sediments studied. Inhibition increased with increasing metal concentration, and amylase was particularly sensitive. Pb2+ generally had the least effect. We conclude that enzyme synthesis measurements are useful in determining the effects of heavy metals on the degradation of organic pollutants in river sediments.

Influence of adrenaline, glucagon, hydrocortisone, thyroxine, or insulin administration on pancreatic exocrine secretion in rats.

Pancreatic exocrine response induced by CCK-PZ (0.2 U/100 g body weight)and amylase content were studied in rats which had been treated with adrenaline, glucagon, hydrocortisone, thyroxine and insulin for 7 days. After insulin or thyroxine treatment, the basal level of pancreatic juice flow and protein output was higher, and the CCK-PZ induced responses were 3-fold larger than those of the corresponding controls. CCK-PZ induced juice flow in hydrocortisone treatment was depressed, but the protein output was not. The ratio of amylase to protein in pancreatic juice and pancreas decreased after adrenaline, glucagon, or thyroxine treatment, but not after insulin or hydrocortisone treatment. Changes in plasma concentration of glucose, free fatty acid and insulin were not correlated directly with a decrease in the amylase content after adrenaline, glucagon or thyroxine treatment. The results indicate the possibility that adrenaline, glucagon, thyroxine and insulin can modify the synthesis and/or release of pancreatic amylase.

Does osmotic regulation of amylase activity in bean cotyledons exist?

The hypothesis that the promotive effect of the embryo axis of the germinating bean seed on amylase activity in the cotyledons is mediated by an osmoregulative mechanism was examined. After 2 days of germination the action of the axis on amylolytic activity was already clearly revealed, whereas at the same time it did not have any influence on osmotic pressure in the cotyledons. When the axis was attached to one cotyledon during 4 days of incubation, osmotic pressure in the cotyledon was lower than its value in the cotyledons of the intact seedling, whereas amylolytic activity was similar in both treatments. It was concluded that the tested hypothesis is not valid in the case of the bean seedling. External osmotic agents brought about a decrease in the level of amylase in the cotyledons, but this does not prove that osmotic changes which are brought about by production of internal metabolites are involved in the regulation of amylase synthesis.

Dietary regulation of pancreatic protein synthesis: II. Kinetics of adaptation of protein synthesis and its effect on enzyme content

The kinetics of the adaptative changes in the relative rates of synthesis and pancreatic concentrations of amylase, chymotrypsinogen and trypsinogen were studied over 10 days of adaptation to a carbohydrate-rich (G), or a protein-rich (P) diet. During adaptation to the P diet, 60% of the adaptative decrease of the amylase to chymotrypsinogen ratio of incorporation was complete within 24 h of feeding and plateau values were obtained after five days. Adaptation to the G diet was only 20% complete after 24 h and plateau values were obtained later than with the P diet. The evolution of the ratio of concentrations of amylase and chymotrypsinogen followed those of incorporation in the adaptation to both diets. These results support the determinant role of adaptative changes in the rates of synthesis of individual enzymes on the dietary adaptation of enzyme proportions in the pancreas. The differences in the kinetics of adaptation to the two diets suggest that different mechanisms are involved in the adaptative regulation of protein synthesis to a carbohydrate-rich diet or a protein-rich diet.

Dietary regulation of pancreatic protein synthesis: I. Rapid and specific modulation of enzyme synthesis by changes in dietary composition

Dietary adaptation of pancreatic protein synthesis and of pancreatic enzyme concentration, was studied over the first 24 h of exposure to a new diet. Rats were adapted to a carbohydrate-rich (G) or to a protein-rich diet (P) and were switched to the opposite regime after a 15 h fast. The evolution of the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen and of the pancreatic concentrations of amylase and chymotrypsinogen were followed. Fasting caused important modifications in the relative rate of synthesis of the three enzymes in rats adapted to a P diet. Adaptative changes in the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen were seen within 2 h after the beginning of refeeding. These changes were followed by corresponding adaptative modifications in pancreatic contents 4 h after the beginning of refeeding. After 24 h of refeeding, significant adaptative changes had occurred in both the relative rates of synthesis and in enzyme concentrations. Thus exocrine pancreatic protein synthesis can be modulated as early as 2 h after refeeding and this modulation is followed by adaptative changes in pancreatic enzyme content.

Regulation of biosynthesis of trypsinogen and chymotrypsinogen by nutritional and hormonal factors in the rat.

Abstract. Experimental diabetes is associated with decreased pancreatic amylase activity resulting from reduced amylase synthesis and increased amounts of trypsinogen and chymotrypsinogen. The effects of different diets and of experimental diabetes on the synthesis of chymotrypsinogen and trypsinogen in rat pancreas were studied by measuring the incorporation of [4,5‐3H]leucine into these proteins by specific im‐munoprecipitation and simultaneous determination of the specific radioactivity of the pancreatic leucine pool. In addition the effect of acute glucagon treatment on the synthesis of these enzymes was examined. In rat pancreas two trypsinogens and one chymotrypsinogen were found. There was no precursor‐product relationship between the two trypsinogens. Changes in the rate of synthesis affected both isoenzymes to the same extent. Starvation was associated with a decreased rate of synthesis of total pancreatic protein, chymotrypsinogen and trypsinogen. A protein‐rich diet led to an increased rate of synthesis of chymotrypsinogen and trypsinogen, but not of total protein. A carbohydrate‐rich diet diminished the amounts of pancreatic chymotrypsinogen and trypsinogen and the rates of synthesis of these enzymes, whereas a fat‐rich diet had no significant effects on these parameters.Diabetes was associated with increased amounts of chymotrypsinogen and trypsinogen in the pancreas. This resulted from a dramatic increase in their rates of synthesis, whereas that of total pancreatic protein showed no significant change. The same pattern was observed in diabetic adrenalectomized rats or diabetic rats receiving the same amount of a standard diet as non‐diabetic controls. Treatment of rats with glucagon for 6 or 12 h led to a marked decrease of the amounts of pancreatic zymogens, whereas the rates of synthesis of chymotrypsinogen and trypsinogen were only slightly diminished.

Immunohistochemical demonstration of the loss of immunoreactive amylase from neoplastic human salivary gland.

Antihuman parotid amylase antibodies raised in rabbits and horses were used as the primary antibodies in both the peroxidase-antiperoxidase and sandwich techniques for the localization of human amylase. Immunoreactive enzyme was demonstrated in the normal acinar cells of salivary glands and pancreas. Malignant transformation, which has occasionally resulted in ectopic production of amylase by various tissues, actually caused a loss of amylase synthesis by the transformed acinar cells of salivary glands and did not result in elaboration of amylase by transformed ductal cells.

Genetic determination of amylase synthesis in the mouse.

Extensive genetic variation in structure and rate of synthesis of pancreatic amylase has been identified among strains of mice. The relative rate of synthesis of amylase varies from 0.15 in strain YBR to 0.26 in strain C3H. The number of electrophoretic isozymes of pancreatic amylase varies between one and four. In each strain with multiple amylase isozymes, a characteristic quantitative distribution of protein among the isozymes is observed. Isozyme proportions are determined by the relative rates of synthesis of each component. Congenic lines with different amylase phenotypes have been established. Genetic analysis reveals the close linkage of cis-acting sites determining rate of synthesis and electrophoretic mobility of mouse pancreatic amylase.

The relation between amylase secreted in vitro by rat parotid tissue and the amylase concentration in the glands

The assumption that the amount of amylase secreted by parotid glands is dependent on the glandular content is often made but seldom substantiated. This association was tested in vitro in parotid tissue. Fasting, feeding and feeding after pre-ganglionic cervical sympathectomy provided contrasting levels of glandular amylase. The subsequent release into fortified Krebs' medium was measured. Secretion was induced by 4 × 10 −7 M carbachol or isoprenaline. Only when the combined results from fasted and fed animals or fed sympathectomized and fed control rats were analysed were the total amounts of amylase released into the medium positively related to the initial amounts in the glands. This applied to spontaneously secreting, carbachol- and isoprenaline-stimulated tissue. When the results from each group were considered separately, this relationship was absent. When the secretion of amylase was expressed as a percentage of the total amylase in the gland, less was released from repleted glands, particularly in those stimulated by isoprenaline. Pre-treatment of tissues in vitro for 4 h with 1.25 × 10 −4 M colchicine impaired amylase release in response to isoprenaline only. Possible explanations for these findings relate to the different distribution and rate of synthesis of amylase in acinar cells of fasted and fed rats.

Effects of ethionine on digestive enzyme synthesis and discharge by mouse pancreas.

The earliest changes noted during the evolution of pancreatitis induced by feeding mice a choline-deficient ethionine-supplemented (CDE) diet are an increase in the number of zymogen granules in pancreatic acinar cells and an increase in digestive enzyme content of the pancreas. We have studied the processes of protein and digestive enzyme synthesis and discharge at varying times after institution of the CDE diet, a choline-deficient diet (CD), and a diet containing ethionine but not choline-deficient (E). Both the CDE and E diets increased digestive enzyme content within 12 h of their institution. Both the CDE and E diets reduced the rate of protein and amylase synthesis and caused a marked reduction in the rate of protein and amylase discharge from the pancreas. These changes were greatest and were noted earliest in the CDE diet group. A marked reduction in secretagogue-induced in vivo and in vitro amylase discharge followed ingestion of either the CDE or E diet. These studies indicate that the increased pancreatic content of digestive enzymes noted after ingestion of the CDE and E diets results from an ethionine-induced decrease in the rat of digestive enzyme discharge. This phenomenon is enhanced by simultaneous choline deficiency. Subsequent intrapancreatic activation of zymogens may couple these changes in enzyme content to the development of hemorrhagic pancreatitis.

Light-mediated amylase synthesis in the petal epidermis of gladiolus

Separation of the outer bract enclosing the flower bud and spike axis results in the stimulation of amylase synthesis in the petals of gladiolus. Light is implicated in this process. Amylase production is localised in the petal epidermis (which is free of starch) and not generalised in the ground parenchyma (which contains abundant starch). Depletion of starch in the petal is strictly basipetal. It is proposed that the reducing sugars released from hydrolysis of starch in the ground parenchyma cause osmotic water intake, leading to petal expansion and flower opening.

Function of Parotid Gland Following Irradiation and Its Relation to Biological Parameters

SASAKI, T., YAMAMOTO, M., AND TAKEDA, M. Function of Parotid Gland following Irradiation and Its Relation to Biological Parameters. Radiat. Res. 83, 579-591 (1980). The function of the parotid gland in the mouse (synthesis and secretion of a-amylase) following X irradiation was analyzed in relation to the parameters of surviving acinar cell fraction, DNA or protein content, and wet weight of the gland. Both synthesis and secretion of amylase in parotid were essentially unchanged when mice were irradiated with a dose of up to 3000 rad. When mice were irradiated and then given a proliferative stimulus of isoproterenol, latent lethal damage in the acinar cell population was expressed and resulted in cell degeneration in a dose-dependent manner. The mean value of amylase activity per gland in similarly treated parotids was, however, totally unaffected. The relationship between amylase activity per gland and the other biological parameters was analyzed by regression analysis. The results indicate that amylase activity per surviving acinar cell increased

Effect of hydrocortisone on pancreatic growth in rats.

This study was undertaken to determine the effects of hydrocortisone on pancreatic size and composition in suckling, recently weaned, growing, and adult rats. The data demonstrate that hydrocortisone 1) is associated with hyperplasia and hypertrophy of the pancreas in suckling rats, 2) suppressed DNA synthesis in recently weaned rats, but maintained the hypertrphic effect, 3) increased protein and pancreatic hydrolase concentrations in growing and adult rats, and 4) preferentially promoted synthesis of amylase over chymotrypsin only during the suckling period. These experiments suggest that growth of the pancreas may be partly under control of glucocorticoids only during the suckling period, whereas hypertrophy of the pancreatic tissue can be obtained at all ages by these hormones.

Effect of sodium chloride on enzyme activity and synthesis in river sediments

Abstract The effect of NaCl, the main component of road de‐icer salt, on amylase, cellulase and urease activity and synthesis in diverse river sediments was determined. Only urease activity was significantly reduced by concentrations of up to 500 μg g−1 Cl− ions. The results suggest that amylase and cellulose breakdown in river sediments is unlikely to be impaired by NaCl, while urea hydrolysis appears more sensitive to Cl concentrations likely to occur in river sediments polluted with de‐icer salt.

Ultrastructural, Cytochemical, and Biochemical Characterization of Alpha-Amylase Produced by Human Gastric Cancer Cells In Vitro

Ectopic production of salivary-type amylase was demonstrated in a human gastric carcinoma cell line (KMK-2) maintained in vitro for more than 3 years. Electron-dense granules, which appeared as zymogens, were observed in the tumor tissue, in cancer cells in the peritoneal fluid (from which the present cell line had been derived), and in cultured cells in early passages. These granules, however, decreased and gradually disappeared during cultivation. Although such a morphologic alteration was recognized, the property of amylase synthesis has been maintained. The presence of alpha-amylase in the cultured cells was demonstrated cytochemically by the immunoperoxidase method. The enzyme secreted and accumulated in the culture medium was partially purified and characterized. Alpha-amylase of KMK-2 cells closely resembled salivary-type amylase in gel filtration profile and disk gel electrophoresis. Immunologic cross-reaction was observed between these enzymes. Secretion into the medium was constant, and the enzyme concentration in the cytoplasm was relatively high when the cells had reached confluence. Prednisolone increased the amylase production two-fold in the cells.

Effect of Moderate Chronic Protein Deficiency on Rat Salivary Components

Chronic protein insufficiency was achieved in rats with an 8% protein diet initiated at weaning. The total activity and activity per milliliter of the principal salivary protein, amylase, were significantly decreased at day 66. The amount of total protein per milliliter of saliva and total protein in stimulated saliva collected during 30 minutes was decreased in the malnourished rats. The levels of immunoglobulin G (IgG) and aminopeptidase in saliva were not affected by dietary status. Prolonged malnutrition on the low-protein, high-starch diet resulted in an apparent stimulation or induction of salivary amylase and protein secretion by day 110. Total protein collected and milligram of protein per milliliter of saliva were very similar in the malnourished and control rats at day 110. Salivary amylase activity in the malnourished rats was significantly greater than in the saliva of controls. The initial decrease in the principal salivary protein, amylase, and in total protein followed by a significant increase with prolonged stimulation by low-protein and high-starch diets suggests that early protein deficiency retards synthesis of amylase and that a high-starch diet may induce salivary amylase production.

Control of Amylase Synthesis in Cotyledons of Germinating Peas: Examination of the Possibility of Osmotic Regulation

Summary Amylase synthesis was severely retarded in detached cotyledons of germinating peas (Pisum sativum L., cv. Alaska 7), while it was markedly promoted in attached ones. The concentration of so-lutes was higher in the former cotyledons than in the latter ones. Even though the solute concentration of attached cotyledons was elevated by addition of external maltose to the level of that of detached ones, amylase synthesis was not inhibited. The enzyme synthesis in attached cotyledons was curtailed only by maltase concentrations that must be regarded as unphysio-logical. These findings are discussed in relation to osmotic regulation of amylase synthesis in pea cotyledons.

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera.

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5-6.0 for E. fibuligera and 5.5-7.0 for S. castellii.

Mycelial Growth and Amylase Production byTalaromyces Emersonii

Extracellular amylase synthesis by Talaromyces emersonii was studied in stationary liquid starch-yeast-extract (SYE) medium. Maximum mycelium and extracellular amylase were produced at 40-45 C, but maximum amylase was produced after autolysis. There was some variation in the relationship between mycelial dry weight and amount of amylase synthesized at the other temperatures tested. Amylase activity was highest at 40-45 C and pH 5-6. Increasing the amount of starch in the medium enhanced mycelial growth and extracellular amylase production. Besides starch, amylase production was also induced when T. emersonii was grown in media containing cellobiose, glucose, fructose, galactose or maltose, but the amount of amylase produced on these carbohydrates was less than when the medium contained starch. Amylase was not produced when sucrose was used as a carbon source.