Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.     

Secretion can proceed uncoupled from net plasma membrane expansion in inositol-starved Saccharomyces cerevisiae

Secretion of acid phosphatase and invertase was examined in an inositol-requiring ino1 mutant of the yeast Saccharomyces cerevisiae. Inositol starvation is known to block plasma membrane expansion, presumably due to restricted membrane phospholipid synthesis. If membrane expansion and extracellular protein secretion are accomplished by the same intracellular transport process, one would expect secretion to fail coordinately with cessation of plasma membrane growth in inositol-starved cells. In glucose-grown, inositol-starved cells, plasma membrane expansion and acid phosphatase secretion stopped coordinately, and intracellular acid phosphatase accumulated. In sucrose-grown, inositol-starved cells, plasma membrane growth halted, but secretion of both acid phosphatase and invertase continued until the onset of inositol-less death. Although glucose-grown and sucrose-grown cells differ in their ability to secrete when deprived of inositol, they exhibited the same disturbances in phospholipid synthesis. Phosphatidylinositol synthesis failed, and its precursors phosphatidic acid and CDP-diglyceride accumulated equally in both cultures. Sucrose-grown yeast cells appear to accomplish normal levels of extracellular protein secretion by an inositol-independent mechanism. In glucose-grown yeasts, both plasma membrane expansion and secretion are inositol dependent.     

Metabolic relationship between invertase and acid phosphatase isoenzymes in Saccharomyces cerevisiae

Repressed cells of Saccharomyces cerevisiae, subjected to inhibition of both RNA and protein synthesis, showed a pattern of membrane-bound and cytosol acid phosphatase to the external enzyme which seemed to be linked through a precursor-product relationship.Gel exclusion chromatography did not indicate clear differences between the isoenzymes. Moreover, centrifugation experiments in CsCl and precipitation with concanavalin A suggested that there were no acid phosphatase molecules devoid of carbohydrate. Membrane-bound invertase displayed a molecular weight and a carbohydrate to protein ratio smaller than those of the exocellular enzyme. The values of molecular weight and buoyant density of the membrane-bound enzyme were closer to those found for the cytosol invertase. The stability of the level of the soluble invertase detected in the cytoplasm under derepression conditions, or after RNA or protein synthesis inhibition was found to be only apparent and represented the result of an equilibrium between synthesis and degradation.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Mutual effect of invertase and acid phosphatase from the yeast Saccharomyces cerevisiae on their secretion into culture media

The hypothesis that various extracellular enzymes produced by the yeast Saccharomyces cerevisiae exert a mutual influence on their secretion into the culture medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.     

Mutual influence of invertase and acid phosphatase of the yeastSaccharomyces cerevisiae on their secretion into the cultivation medium

The hypothesis that various extracellular enzymes produced by the yeastSaccharomyces cerevisiae exert a mutual influence on their secretion into the cultivation medium was tested experimentally. The statistically processed results indicate that extracellular invertase affects the secretion of acid phosphatase, and acid phosphatase affects the secretion of invertase. In addition, the secretion of each of these enzymes was shown to be subject to autoregulation.     

Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.     

Recovery of exocellular acid phosphatase activity on Saccharomyces mellis after treatment of the organism with reagents that affect the cell surface

Derepressed cells of Saccharomyces mellis were treated in one of several different ways to either elute or inactivate the exocellular enzyme, acid phosphatase. The enzyme was either (i) eluted from resting cells with 0.5 m KCl plus 0.1% beta-mercaptoethanol, (ii) eluted from exponential phase cells by growing the organism in derepressing media containing 0.5 m KCl, or (iii) inactivated on exponential phase cells by adding sufficient acid or base to growth media to destroy the enzyme but not enough to kill the cells. These treatments did not affect viability. Treated cells were transferred to fresh growth media or some other reaction mixture, and the kinetics of recovery of acid phosphatase activity was studied. In these reaction mixtures, enzyme was synthesized only by actively growing cells. Treated resting cells were indistinguishable from untreated, repressed resting cells in that the organism inoculated into complete growth medium remained in the lag phase for approximately 6 hr before both growth and enzyme synthesis began. Exponential phase derepressed cells treated by method (ii) or (iii) were transferred to fresh medium under conditions that allowed growth to continue. The cells immediately started to manufacture enzyme at a rate greater than normal until the steady-state level was reached, thus demonstrating a feedback control system. Exponential phase repressed cells were also transferred to fresh derepressing media under conditions which sustained growth. Though these cells began to grow immediately, there was a lag before acid phosphatase synthesis began followed by a lengthy inductive period. The length of the period of induction could be correlated with the polyphosphate content of the cells. As the supply of polyphosphate neared exhaustion, the rate of synthesis increased rapidly until it was greater than normal; this differential rate was sustained until the steady-state concentration was reached. When derepressed cells grow in a medium containing 0.5 m KCl, some acid phosphatase activity is found free in the culture fluid and some remains firmly attached to the cells despite the presence of the salt. The bound activity is subject to feedback control, but the steady-state level of this activity on the cells is only one-third that of the acid phosphatase on cells growing in nonsaline media. The extracellular phosphatase is produced at a rate that is several-fold greater than that of the exocellular enzyme in a nonsaline medium. The synthesis of the extracellular enzyme does not seem to be controlled by a feedback mechanism but is produced at a maximal rate as long as the cells are growing.     

Synthesis of beta-glucanase and other extracellular proteins by cells and protoplasts of the yeast Pichia polymorpha: effect of 2-deoxy-D-glucose.

The synthesis of beta-glucanase either by cells or by protoplasts of the yeast Pichia polymorpha has been found to occur in the presence of 2-deoxy-D-glucose in the growth medium. On the other hand, the synthesis of typical extracellular proteins such as invertase and acid phosphatase is strongly affected by the presence of the drug. The degree of inhibition is, however, directly related to the 2-deoxy-D-glucose concentration.     

Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane

Summary Using sugar-cane internodal tissue in which RNA synthesis was ratelimiting for invertase of peroxidase synthesis, measurements were made of enzymeforming-capacity after blocking further RNA synthesis with actinomycin D or 6-methylpurine. In this way it was possible to determine whether added auxin (naphthaleneacetic acid) or gibberellic acid (GA₃) affected steps prior or subsequent to synthesis of the RNA fractions specifically required for synthesis of either enzyme. Both auxin and GA₃ increased the enzyme-forming-capacity for invertase but not for peroxidase. The effects of the two hormones are interpreted as causing stabilization of mRNA for invertase.Abscisic acid (ABA) increased the rate of synthesis of invertase but not peroxidase. ABA did not change the rate of loss of invertase when peptide-bond formation was blocked with cycloheximide, but stimulated its synthesis when RNA synthesis was blocked with 6-methyl purine. Hence, the site of action of ABA is subsequent to invertase-mRNA formation and prior to invertase destruction.Kinetin had no short-term effects when RNA synthesis was limiting for invertase production, and does not appear to directly modulate mRNA synthesis or stabilization, or amino-acid-polymerization steps. In treatments longer than 5 hours, kinetin inhibited synthesis of all three enzymes studied, so that its effect on enzyme synthesis in this tissue appears to be unspecific.Abbreviations used throughout text ABA (±)-abscisic acid (abscisin II, dormin) - GA₃ gibberellic acid - NAA -naphthaleneacetic acid     

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.     

Acid phosphatase activity and microcyst differentiation in the cellular slime mold Polysphondylium pallidum

The enzyme acid phosphatase (E.G. 3.1.3.2) is present in Polysphondylium pallidum and increases during microcyst differentiation. The increase of enzyme activity occurs during the period of morphological differentiation of microcysts. The use of the inhibitor cycloheximide provides evidence that the increase in enzyme activity requires coincident protein synthesis. Acid phosphatase activity also accumulates extracellularly but the extracellular release of this activity is not stopped by cycloheximide. A cystless mutant (strain NG-6) shows a high but essentially unchanging intracellular level of acid phosphatase and a slightly delayed extracellular pattern of accumulation. A comparison of other enzyme patterns in strain NG-6 indicates that different control mechanisms in addition to the regulation of translation may mediate the appearance of. different enzymes during microcyst differentiation.     

潮棕壤免耕农田土壤酶活性的动态变化

研究了潮棕壤免耕和常规耕作农田土壤蔗糖酶、脲酶和酸性磷酸酶活性在玉米不同生育时期和不同土层深度的动态变化.结果表明,免耕可显著提高表层(0～10 cm)土壤酶活性,其蔗糖酶活性在玉米拔节期、大喇叭口期和成熟期显著高于常规耕作,脲酶活性在拔节期和孕穗期显著高于常规耕作,酸性磷酸酶活性在孕穗期和成熟期显著高于常规耕作(P＜0.05);在10～20cm土层,免耕土壤蔗糖酶活性在苗期、拔节期和大喇叭口期与常规耕作差异显著,脲酶活性除孕穗期外均显著高于常规耕作(P＜0.05);在20～30 cm土层,免耕土壤蔗糖酶活性在玉米各生育期均显著低于常规耕作,土壤脲酶活性在苗期、酸性磷酸酶活性在成熟期与常规耕作差异显著(P＜0.05).随土层深度的增加,免耕农田土壤酶活性总体呈下降趋势;常规耕作农田土壤蔗糖酶和酸性磷酸酶活性总体呈上升趋势,而脲酶活性呈下降趋势.     

Regulation of invertase in Aspergillus nidulans: effect of different carbon sources

Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.     

Effects of cycloheximide, actinomycin D and ethylene on the increase and subsequent decrease in acid invertase activity in wounded sweet potato

In wounded sweet potato root tissue, the mechanisms of the increaseand subsequent decrease of acid invertase activity were examinedin compariosn with phenylalanine ammonia lyase (PAL) and peroxidaseactivities. The increase in acid invertase and PAL activitiesin response to wounding was inhibited by both cycloheximideand actinomycin D. However, the increase in peroxidase activitywas inhibited by cycloheximide but was not inhibited by actinomycinD, which repressed RNA synthesis 60 to 70%. The increase inacid invertase activity was supressed by ethylene, while PALand peroxidase activities were stimulated. The results of cycloheximideco-treatment suggest that the fluctuation in enzyme activitiesby ethylene treatment involves de nove protein synthesis. Further,the effect of ethylene on enzyme activities was under competitionby carbon dioxide. The decrease in acid invertase activity atthe late stage of incubation was stimulated by cycloheximidetreatment, but that of PAL activty was prevented. The resultssuggest the existence of different inactivating systems betweenacid invertase and PAL. ¹ This paper constitutes Part 117 in the Series "The PhytopathologicalChemistry of Sweet Potato with Black Rot and Injury". (Received September 2, 1974; )     

The distribution of acid invertase in developing leaves of Lolium temulentum L.

The levels of invertase (E.C. 3.2.1.26) activity were measured throughout the development of the fourth leaf of Lolium temulentum. No neutral invertase activity was detected. Soluble acid invertase activity fell during leaf extension but rose again after ligule formation. This rise continued into senescence and was accompanied by the appearance of activity in the insoluble fraction. Evidence is presented that the insoluble activity was not an artefact of preparation, and that it represented an extracellular acid invertase. Fractionation of soluble invertase by gel filtration showed the appearance of a high molecular weight form at the time when insoluble activity was rising. The relationships between the different forms of the enzyme are discussed, together with their roles in leaf development.     

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces was examined using sorbitol, KCl, NaCl, or magnesium sulfate as the osmotic support. The synthesis and secretion of the enzyme is remarkably sensitive to the osmolarity of the supporting medium irrespective of the particular support employed. Invertase formation was inhibited at high osmolarity and was maximal at 0.65 to 0.75 osmolal, even though some leakage of the intracellular enzyme alpha-glucosidase and of ultraviolet (UV)-absorbing materials occurred under these conditions. The reduction of invertase formation and secretion due to high osmolarity was eliminated promptly when protoplasts were transferred into a medium of lower osmolarity. The rate of fructose uptake and of threonine incorporation into protein was decreased by high osmolarity; also reduction of invertase formation could be partially reversed by increasing the level of sugar supplied as energy source. Thus changes in the permeability of the plasma membrane (and presumably also in its structure) are important factors in the response of protoplasts to high osmolarity, though certainly not the complete explanation. Protoplasts suspended in 0.8 m sorbitol, with 10mm fructose as the energy source, increased their invertase level 5- to 10-fold during a 2-hr incubation without substantial release of alpha-glucosidase or UV-absorbing materials. Both the large and small forms of invertase were present in the protoplasts, but only the large form was released into the medium when enzyme was being actively synthesized. Formation and secretion of newly formed invertase and the release of enzyme initially present were inhibited by cycloheximide.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA : I. Effect of Glucose, Acetate, Pyruvate, and Carmine on Intracellular Content and Extracellular Release of Three Acid Hydrolases

Log-phase Tetrahymena were washed and resuspended in a dilute salt solution supplemented with glucose, acetate, pyruvate, or carmine, as desired, and then incubated for 5 h. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed. Extracellular activities were corrected for proteolytic degradation. The three nutritive substrates affected both the amount and pattern of extracellular enzyme release, but carmine had no effect. Intracellular activities declined early in the starvation period, but partially recovered with time, particularly α-glucosidase activity. Acetate reduced the decline in acid phosphatase activity; acetate and glucose enhanced the recovery of α-glucosidase activity; carmine had no effect on intracellular enzyme activities. Protein content changed little and was unaffected by the addition of substrates. Glycogen content increased during incubation; acetate and glucose enhanced the increase.     

Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation.

Photoautotrophic suspension-culture cells of Chenopodium rubrum that were shifted to mixotrophic growth by adding glucose were used as model system to investigate the influence of the source-sink transition in higher plants on the expression and enzyme activities of intracellular and extracellular invertases. The complete cDNA coding for an extracellular invertase was cloned and sequenced from C. rubrum, and its identity has been proven by heterologous expression in Saccharomyces cerevisiae. The higher activity of extracellular invertase after preincubation in the presence of glucose was paralleled by an increased expression of the corresponding gene. The induction by glucose could be mimicked by the nonmetabolizable glucose analog 6-deoxyglucose. Both enzyme activity and mRNA level of extracellular invertase showed a sink-tissue-specific distribution in plants. The activity of neutral and acidic intracellular invertases were not affected by preincubation of autotrophic tissue cultures with sugars, nor did they show a tissue-specific distribution in plants. The data suggest that apoplastic invertase not only has an important function in phloem unloading and carbohydrate partitioning between source and sink tissues but may also have a role in establishing metabolic sinks.