Kinetics of enzyme release from breadmaking yeast cells in a bead mill

Summary Four intracellular enzymes from two species of breadmaking yeasts- S. cerevisiae and C. boidinii- have been measured as a function of time during its disruption using a bead mill in batch operation. The amount and rate of enzyme released was dependent on its location inside the cell as well as on the kind of yeast. The maximum amount of invertase, a-D-glucosidase, alcohol dehydrogenase and fumarase was obtained at 2,5,10,15 min. respectively for S. cerevisiae. C. boidinii did not show either invertase nor a-D glucosidase activity and the maximum amount of alcohol dehydrogenase and fumarase were reached at 5 and 20 min. respectively.     

MODIFICATION OF ZYMOSAN-INDUCED RELEASE OF LYSOSOMAL ENZYMES FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES BY CYTOCHALASIN B

During the process of phagocytosis, polymorphonuclear leukocytes (PMN) release lysosomal enzymes into the extracellular medium. When the antibiotic cytochalasin B (CB) is present in the incubation medium along with phagocytable particles, enhanced recovery of enzyme activities from the incubation medium has been observed. These findings have led to the interpretation that CB enhances lysosomal enzyme release. Our results contradict this interpretation. The lysosomal enzymes acid phosphatase and β-galactosidase are unstable after they are released from cells. During the first 5–15 min of phagocytosis, significant amounts of both acid phosphatase and β-galactosidase can be recovered from the extracellular medium. After this, the recovery of enzyme from the medium declines, presumably because the rate of loss of lysosomal enzyme activity exceeds the rate of release at later time periods. In the presence of CB, the appearance of lysosomal enzymes in the extracellular medium of cells exposed to zymosan is retarded for 5–10 min, after which it begins and then continues for approximately 20 min. At the end of a 30-min incubation period, therefore, in the absence of CB, extracellular levels of lysosomal enzymes (especially those which are unstable) are declining toward low levels while, in the presence of CB, extracellular enzyme levels are continuing to rise. We also measured the lysosomal enzyme remaining within cells after exposure to zymosan. CB retarded the disappearance of enzyme from cells and resulted in significantly less total cell enzyme loss. Thus, in the presence of CB, a greater proportion of the lysosomal enzyme lost from cells is recovered in the extracellular medium. In contrast to the previous conclusions that CB enhances lysosomal enzyme release, our results indicate that CB delays and decreases the zymosan-stimulated release of lysosomal enzymes from PMN. Since CB inhibits phagocytosis by PMN, our results indicate that the antibiotic modifies the mechanism of release of lysosomal enzymes, resulting in zymosan stimulation of their release independently of phagocytosis.     

Differential product release (DPR) of proteins from yeast: a new technique for selective product recovery from microbial cells

A novel method has been developed for the separation of bioproducts from yeast cells. The method uses a combination of physical, chemical, and biological agents such as lytic enzymes, osmotic supports, and spheroplast stabilizers. Using this technique, products (proteins and enzymes) can be released from specific cell locations at different process states; it has thus been celled differential product release (DPR). The wall-associated proteins are released first and the lytic enzyme is removed together with the wall proteins at this stage. Secondly, the cytosol products are released by a mild procedure during which the organelles remained intact. Finally, the organelle proteins are solubilized. In each stage, specific proteins are released while others are kept inside the different cell compartments. This method can be used with relatively high yeast concentrations (up to 145 g dry wt/L) and gives higher product recoveries and much higher selectivity than mechanical disruption.     

Statistically designed optimisation of enzyme catalysed starch removal from potato pulp

Potato pulp is a high-volume, low-value byproduct stream resulting from the industrial manufacture of potato starch. The pulp is a rich source of biologically functional dietary fibers, but the targeted valorisation of the fibers requires removal of the residual starch from the pulp. The objective of this study was to release the residual starch, making up 21–22% by weight of the dry matter, from the potato pulp in a rational way employing as few steps, as few enzyme activities, as low enzyme dosages, as low energy input (temperature and time), and as high pulp dry matter as possible. Starch removal to obtain dietary fibers is usually accomplished via a three step, sequential enzymatic treatment procedure using a heat stable α-amylase, protease, and amyloglucosidase. Statistically designed experiments were performed to investigate the influence of enzyme dose, amount of dry matter, incubation time and temperature on the amount of starch released from the potato pulp. The data demonstrated that all the starch could be released from potato pulp in one step when 8% (w/w) dry potato pulp was treated with 0.2% (v/w) (enzyme/substrate (E/S)) of a thermostable Bacillus licheniformis α-amylase (Termamyl^® SC) at 70 °C for at least 65 min. The study also indicated that the amount of other carbohydrates released from the pulp during the release of starch was less than using the AOAC Official Method 985.29 and another recently published starch release method employed as a pretreatment for enzymatic upgrading of a pectinaceous potato pulp fiber.     

Extramitochondrial localization of NADH-fumarate reductase in trypanosomatids

Trypanosoma brucei procyclic trypomastigotes and T. cruzi epimastigotes (both Tulahuen and Y strains) were permeabilized by incubation with increasing amounts of digitonin, causing enzymes to be released from different intracellular compartments. After 10 min incubation with digitonin, the cells were centrifuged and the activity of marker enzymes (aspartate-dependent malic enzyme for cytoplasm, hexokinase for glycosomes and either isocitrate dehydrogenase or citrate synthase for mitochondria) was analyzed in the supernatant. The results were compared with the release of NADH-fumarate reductase in order to determine if this enzyme was preferentially released with a specific intracellular marker. Fumarate reductase was released at lower digitonin concentration than those required to either release isocitrate dehydrogenase or citrate synthase. Similarly, Leishmania donovani promastigotes (S-2 strain) were exposed to a single concentration of digitonin (200 micro M) but in this case we monitored the release of fumarate reductase and hexokinase, while monitoring the mitochondrial membrane potential (using safranine O). Again, substantial fumarate reductase and hexokinase activities were released without loss of mitochondrial membrane potential indicating that part of the enzyme was released while the inner mitochondrial membrane remained intact. These results suggest that, in the three species of trypanosomatids the enzyme fumarate reductase is, at least in part, located outside the mitochondrial matrix.     

Adenylate cyclase activity in permeabilized cells from Dictyostelium discoideum

Aggregation competent cells of Dictyostelium discoideum have been permeabilized using the method previously developed for mammalian cells by Aragón et al. (Proc. Natl. Acad. Sci. USA (1980) 77, 6324). The permeabilization allows the “in situ” assay of adenylate cyclase. The enzyme activity is proportional with the amount of cells and the time of incubation. The enzyme exhibits non Michaelian kinetics towards its substrate ATP as recently reported for the enzyme assayed in crude extracts. Permeabilized cells contain lower levels of phosphodies-terase activity as compared with cellular homogenates. The similar properties of the adenylate cyclase from cell homogenates and permeabilized cells, as well as the low phosphodiesterase activity, makes the permeabilization method of great interest to study the regulation of adenylate cyclase during the differentiation of Didiscoideum.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

Release of hyaluronidase and beta-N-acetylhexosaminidase during in vitro incubation of hamster sperm

Previous studies have shown that hamster sperm release a significant amount of hyaluronidase before and independently of the normal acrosome reaction. In this study, we have used improved methods for in vitro incubation to investigate the time course of the release of hyaluronidase and hexosaminidase from hamster sperm. When hamster sperm are incubated in medium which allows capacitation, 34 to 47% of the total mechanically extractable hyaluronidase and 34 to 51% of beta-N-acetylhexosaminidase are released into solution prior to and independently of the normal acrosome reaction (ARx). An additional 40 to 50% of the hyaluronidase and 34 to 51% of the hexosaminidase are released at the time of the normal ARx. Control experiments indicate that the early release is not due to the presence of dead sperm in culture and that the normal ARx is required for the second release. Increasing amounts of TCA-precipitated bovine serum albumin in the culture medium stimulated the early (1 hr) release of both enzymes. The data are consistent with the ideas that a significant amount of both enzymes is released from the sperm surface by 1 hr of incubation and that about the same amount of each enzyme is released during the normal ARx. Hyaluronidase and hexosaminidase release at the time of the acrosome reaction was measured for the first time using hamster sperm. The biphasic release of these enzymes may indicate that they have a dual function in fertilization and may help explain how sperm can penetrate the cumulus and corona radiata without undergoing an acrosome reaction.     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Proteoglycan-degrading enzymes. A radiochemical assay method and the detection of a new enzyme cathepsin F.

1. Polyacrylamide beads containing entrapped 35S-labelled proteoglycan molecules have been prepared. 2. The measurement of release of radioactivity provides an extremely sensitive assay for proteoglycan-degrading enzymes, including proteinases and hyaluronidase. 3. The amount of label released is a logarithmic function of enzyme concentration or time of incubation. Experiments were made in an attempt to explain this. 4. Assays were made by the new method at several pH values, and with the inclusion of inhibitors to identify the proteoglycan-degrading enzymes of rabbit ear cartilage. 5. A previously undescribed proteinase active against proteoglycan at pH4.5 but unaffected by pepstatin, was discovered. The enzyme was named cathepsin F, and was partially purified and characterized; it was detected in human articular cartilage.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

Summary The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasocative peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8–13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response.

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.     

THE MOBILIZATION AND EXTRACELLULAR RELEASE OF GRANULAR ENZYMES FROM HUMAN LEUKOCYTES DURING PHAGOCYTOSIS

The importance of granular (lysosomal) enzymes from neutrophils in producing the tissue damage of acute inflammation has been suggested by much indirect and some direct evidence. This study has investigated the kinetics of release and subsequent fate of granular enzymes from phagocytizing human leukocytes The following observations are made: (a) During phagocytosis, the granular enzyme lysozyme is released from leukocytes into the extracellular medium. (b) Release of lysozyme increases as phagocytic challenge increases, but attains a maximum. (c) Release of lysozyme accompanies phagocytosis and is not a delayed event. (d) The lack of release of a nongranular enzyme, lactic dehydrogenase, indicates that cell damage is not a necessary condition of enzyme release. (e) Like lysozyme, β-glucuronidase is released from phagocytizing leukocytes. Acid α-naphthyl phosphatase and cathepsin also appear to be released, but are not found in appreciable amounts in the extracellular medium, in part because of their lability in solution. These results support the concept that extracellular release of granular enzymes may be a useful secretory function of inflammatory leukocytes which becomes damaging to the host in certain circumstances.     

Secretion of β-glucosidase by Ochromonas danica

-Glucosidase released by the phytoflagellate Ochromonas danica was the result of secretion; this was adduced from the following: (1) The enzyme was released during growth, including early log phase. (2) The amount released was calculated to be much more than could be attributed to cell lysis. (3) -Glucosidase was released by cells during short term incubation in a dilute salt solution; this release was nearly linear for at least 24 h. (4) Release occurred while cell counts remained nearly constant and cells remained viable. (5) Control experiments excluded cell damage resulting from incubation and cell manipulation as a source of the exoenzyme. (6) No alkaline phosphatase was released and 5 times less phosphoglucose isomerase than glucosidase was released while the cells contained 7 times more phosphoglucose isomerase. (7) The kinetics of release of nonspecific protein and UV absorbing material was markedly different from glucosidase release. (8) Glucosidase release was temperature and energy dependent; anaerobiosis decreased enzyme release. (9) Release was inhibited by cycloheximide. (10) Cells incubated with ³H-leucine synthesized labeled protein which was secreted linearly for at least 24h. Cycloheximide inhibited incorporation of ³H-leucine into protein and the secretion of the labeled protein.Non-Standard Abbreviations CHI cycloheximide - DNP 2,4-dinitrophenol - IAA iodoacetic acid - PGI phosphoglucose isomerase - SIS salt incubation solution     

Kinetic aspects of enzyme activity changes in blood plasma during canine hemorrhagic shock

The kinetics of the increase in activities of eight enzymes in plasma was investigated in hemorrhagic shock in dogs (8.0 kPa, 120 min). The time-course of enzyme activity changes in shock differed between animals and depended on their sensitivity to shock. In the shock-sensitive group of dogs an exponential activity increase was already observed in the hypotension period. However, the dogs of the less shock-sensitive group showed a delay of enzyme release with significantly less pronounced elevation of all enzyme activities except creatine kinase. The initial exponential rise of enzyme activities, which approximately followed first-order kinetics, was quantitatively characterized by the release rates. There was a close correlation between the molecular weights of enzymes and their release rates during shock in both groups of dogs. The relevance of the results to mechanisms of enzyme transport from the cell into the blood is discussed.     

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.     

Competence-related increased enzyme release from Streptococcus sanguis (Wicky) cells.

The ablity of competent and noncompetent Streptococcus sanguis (strain Wicky) cells to release enzymes to the environment was studied. Both competent and noncompetent cells leaked the enzymes tested (aldolase, phosphatase and deoxyribonuclease), but the activities liberated from the competent cells were always roughly 2-fold higher than those released from noncompetent cells. This increased enzyme leakage from competent cells occured in all kinds of media and procedures employed. The leakage of enzymes followed a time-dependent kinetics (different for aldolase and phosphatase), was temperature sensitive and had a pH optimum. The increased enzyme release was most likely not due to cell disruption, but seemed to be rather a consequence of alteration in cell barrier permeability. These results strongly support the "unmasking" model proposed for explanation of competence development in bacteria.     

Production of 2,3-butanediol from HF-hydrolyzed aspen wood

Hydrolyzates from hydrogen fluoride (HF) treated aspenwood were predominantly composed of oligosaccharides which are not readily utilized by Klebsiella pneumoniae. Attempts at further hydrolyzing these oligosaccharides using a variety of glycolytic and xylanolytic enzymes (i.e., amylases, cellulases, and xylanases) were only partially successful. When a post-hydrolysis step was carried out using 3% H₂SO₄, significant amounts of the component monosaccharides were detected. Sugars released by acid or enzymatic hydrolysis of the HF treated aspenwood were utilized by K. pneumoniae for the production of butanediol and ethanol.     

Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis

Cultured chromaffin cells can be permeabilized with digitonin; the cell interior is then accessible to the cytoplasm, and addition of calcium provokes release of catecholamines. Increasing the incubation time between the permeabilization step and calcium-induced stimulation resulted in a progressive inhibition of secretion reaching 60% after 20 min. Cytosoluble proteins which leak from detergent-permeabilized cells were collected, dialyzed, and concentrated. When these proteins were added back to permeabilized cells which were unable to secrete, catecholamine release was fully restored, suggesting that certain proteins necessary for exocytosis had been dialyzed from these cells. One of the released proteins was characterized as calmodulin. However, addition of calmodulin alone was ineffective in maintaining or restoring secretory activity in digitonin-permeabilized cells, excluding calmodulin as the sole factor responsible for the loss of release. Protein kinase C was also identified as one of the leaked proteins. This enzyme is known to be retained in cells in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). However, under TPA-dependent conditions, there was also a loss of secretory activity. The present paper shows that among the proteins leaked from digitonin-permeabilized cells, there are specific proteins crucial to the exocytotic mechanism.