Location of Acid Phosphatase and β-Fructofuranosidase Within Yeast Cell Envelopes

After 16 hr of incubation in a low-phosphate, aerated medium, bakers' yeast was obtained with a high titer of acid phosphatase (EC 3.1.3.2) and beta-fructofuranosidase (EC 3.2.1.26). All of the beta-fructofuranosidase and 75% of the acid phosphatase were easily released by mechanical disruption in a French pressure cell. The cell wall suffered a limited number of cracks, but this was sufficient for the co-release of these enzymes. Both enzymes were subject to autolytic release, although correlation was inconclusive because of the relative instability of acid phosphatase. The data are consistent with the bulk of the two enzymes being located in the periplasmic space. Ethylacetate treatments yielded ghosts with high beta-fructofuranosidase but low acid phosphatase activities. The surviving acid phosphatase was not representative of that in live cells. It was resistant to release by mechanical disruption and showed a high susceptibility to heat inactivation. The beta-fructofuranosidase in live cells and in ethylacetatetreated cells exhibited polydispersity in heat inactivation susceptibility; but the kinetics were indistinguishable, and facile release by mechanical disruption was shown in both cases.     

Effects of phospholipases C on membrane-bound enzymes of yeast

Alkaline phosphatase was released from protoplasts of the yeast Saccharomyces cerevisiae without cell lysis not only by phosphatidylinositol (PI)-specific phospholipase C but also by phosphatidylcholine (PC)-hydrolyzing phospholipase C. Activities of mitochondrial enzymes such as succinate dehydrogenase, antimycin-sensitive NADH-cytochrome c reductase, and oligomycin-sensitive ATPase were decreased by the action of PC-hydrolyzing phospholipase C. Hydrolysis of microsomal PC or PI did not cause any decrease in the activities of NADPH-cytochrome c reductase and antimycin-insensitive NADPH-cytochrome c reductase. In the requirement of phospholipids, the properties of yeast mitochondrial enzymes were very close to those of mammalian mitochondrial enzymes, whereas those of yeast microsomal enzymes were completely different from those of mammalian microsomal enzymes.     

Lysosomal Physiology in Tetrahymena. II. Effect of Culture Age and Temperature on the Extracellular Release of 3 Acid Hydrolases*

Tetrahymena cultures were grown from a single inoculum and collected on 3 successive days corresponding to the log, transitional, and early stationary phases of growth. Cells were washed and incubated for 5 hr in a dilute salt solution. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed, and extracellular activities corrected for proteolytic degradation. A marked increase in the cellular content of acid phosphatase and significant decreases in α-glucosidase and ribonuclease occurred with advancing culture age. The intracellular changes in enzyme activities during incubation were roughly similar for cells of all ages. Protein content did not change appreciably during incubation. Extracellular A₂₅₅ release, monitored as an indication of the loss of RNA breakdown products, was at a minimum during incubation of transition cells. Significant quantities of all 3 acid hydrolases were released from cells of all ages except for ribonuclease from transition cells. The release of acid phosphatase and α-glucosidase was approximately proportional to the initial cellular content of these enzymes for cells of different ages and in log cells the effect of temperature on the rates of release was described by the Arrhenius equation. Release of ribonuclease, however, was not proportional to its intracellular content nor did it vary with temperature according to the Arrhenius equation. The results suggest that acid phosphatase and α-glucosidase are released via a first-order process.     

Comparative release of alkaline phosphatases from Serratia marcescens by osmotic shock and polymyxin B treatment.

The comparative release of periplasmic enzymes and proteins from two strains of Serratia marcescens by osmotic shock and polymyxin B treatment was studied. There were significant qualitative and quantitative differences in the materials released by these two techniques. The osmotic shock procedure released a higher level of alkaline phosphatase activity and a greater number of protein components than the polymyxin B treatment. The molecular weights of the active components released by the two techniques were shown to be 190,000 +/- 10,000 (A'), 140,000 +/- 10,000 (A) and 110,000 +/- 10,000 (B) daltons. Components released by polymyxin B were also released by osmotic shock. However, the reverse was not true. Component B in the osmotic shock fluids was by far the most active. The differences in the release mechanisms of the two techniques were discussed. It is suggested that polymyxin B treatment is the method of choice because of its selectiveness and mildness, despite the rather low level of activity of alkaline phosphatase released.     

Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.

The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.     

Characterization of orthophosphate release from dissolved organic phosphorus by gel filtration and several hydrolytic enzymes

The molecular weight distribution of dissolved organic phosphorus (DOP) and the possible mechanisms of orthophosphate (Pi) release were examined by gel filtration and incubation with some hydrolytic enzymes. Sixty five percent of the DOP appeared to have apparent molecular weights between 300 to 10000 daltons. Less than 10% of the DOP estimated higher molecules greater than 10000 daltons. Alkaline phosphatase released Pi more easily from low molecular weight (< 1500 daltons) DOP than from high molecular weight fractions. While, addition of nucleases or phosphodiesterase alone did not appear Pi release from high molecular weight DOP compounds. Pi release from those DOP compounds increased markedly (more than 30%) when alkaline phosphatase was incubated with nucleases or phosphodiesterase. However, 60% of DOP did not release Pi when alkaline phosphatase was incubated with either enzymes.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.     

Effect of cryogenic preservation (−80 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

The effect of cryopreservation on plasma membrane and granule associated enzymes of polymorphonuclear neutrophils (PMNs) was studied. The activity of PMNs to generate superoxide anions during phagocytosis was very sensitive to cryopreservation and exhibited approximately 60% inhibition in 24 hr. The total enzyme activity was not as affected during 1-month cryopreservation as that observed with the extracellular release of enzymes. Acid p-nitrophenyl phosphatase and peroxidase were released slightly from frozen and thawed PMNs. However, the extracellular release of LDH, a cytosol marker, and β-glucuronidase and lysozyme, granuleassociated enzymes, increased with cryopreservation time. The degree of release of these enzymes was LDH > β-glucuronidase > lysozyme. A considerable amount of LDH was extracellularly released after 1-month storage. Frozen and thawed PMNs became sensitive to hypotonic solutions, although fresh, nonfrozen PMNs were very resistant to hypotonic lysis. The hypotonic fragility increased even after 1 hr of cryopreservation.Addition of ATP to the preservation medium did not improve enzyme activity, enzyme release, or stimulated superoxide anion generation but increased the hypotonic fragility of PMNs. However, albumin showed protective effects against cryopreservation injury to the O₂^?-generating system, the extracellular enzyme release, and osmotic fragility.     

Ecto-protein kinase release differs from cleavage by phospholipases of a glycosyl-phosphatidylinositol membrane anchor.

Ecto-protein kinases have been detected as physiological constituents of cells. One feature of ecto-phosvitin/casein kinase (ecto-PK) is its release from the surface in a soluble form when cells are incubated with exogenous substrate protein. This is interesting in view of the fact that some ecto-enzymes are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Such enzymes are known to be released from the surface through cleavage by a phospholipase activity. We therefore investigated whether bacterial phospholipase C (PI-PLC) was able to release ecto-PK from intact HeLa cells. The data show that whereas alkaline phosphatase, known to be GPI-anchored, was solubilized, the ecto-PK was neither released nor affected in its activity. Another effect of treatment of cells with phospholipases was the formation of diacylglycerol or phosphatidic acid which, however, did not occur when cells were incubated with phosvitin, the condition which induces ecto-PK release. These results coherently indicate that cellular phospholipases are not involved in the release mechanism of ecto-PK. Also, the presence of various protease inhibitors did not affect ecto-PK release. Cross-linking of cell-surface proteins by bifunctional agents of the succinimidyl-type suggest a protein-protein interaction responsible for membrane anchoring of the ecto-PK.     

Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C.

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on L-leucyl-beta-naphthylamidase, alkaline phosphodeisterase I and Ca2+- or MG2+-ATPase, but substantial proportions of the alkaline phosphatase and 5-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not exluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.     

Activity of protein phosphatases against initiation factor-2 and elongation factor-2.

The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.     

Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins.

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, omega-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose. The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-alpha-D-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-alpha-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-alpha-maltotriosyl cyclomaltoheptaose and 6-O-alpha-maltotetraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase. 6-O-alpha-D-Glucosyl cyclomaltoheptaose and 6-O-alpha-D-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-alpha-maltotriosyl cyclomaltoheptaose as from 6-O-alpha-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin. The yeast debranching enzyme appears to be exclusively oligo-1,4----1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.     

Effects of bile salts on the plasma membranes of isolated rat hepatocytes.

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.     

Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium.

The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.     

ON THE MECHANISMS OF BONE RESORPTION : The Action of Parathyroid Hormone on the Excretion and Synthesis of Lysosomal Enzymes and on the Extracellular Release of Acid by Bone Cells

Bone resorption, characterized by the solubilization of both the mineral and the organic components of the osseous matrix, was obtained in tissue culture under the action of parathyroid hormone (PTH). It was accompanied by the excretion of six lysosomal acid hydrolases, which was in good correlation with the progress of the resorption evaluated by the release of phosphate, calcium 45 or hydroxyproline from the explants; there was no increased excretion of two nonlysosomal enzymes, alkaline phosphatase, and catalase. Balance studies and experiments with inhibitors of protein synthesis indicated that the intracellular stores of the acid hydrolases excreted were maintained by new synthesis. The release was not due to a direct disruption of the lysosomal membrane by PTH; it is presumed to result from an exocytosis of the whole lysosomal content and to involve mechanisms similar to those controlling the secretion of this content into digestive vacuoles. The resorbing explants acidified their culture fluids at a faster rate and released more lactate and citrate than the controls; this release was in good correlation, in the PTH-treated cultures, with the resorption of the bone mineral, but the amount of citrate released was considerably smaller than that of lactate. The acid released could account for the resorption of the mineral. It is proposed, as a working hypothesis, that the acid hydrolases of the lysosomes are active in the resorption of the organic matrix of bone and that acid, originating possibly from the stimulation of glycolysis, cares for the concomitant solubilization of bone mineral while also favoring the hydrolytic action of the lysosomal enzymes.     

Alkaline phosphatase in HeLa cells. Stimulation by phospholipase A2 and lysophosphatidylcholine.

Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent Km. The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures

Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations. Correspondence to: C. Nombela     

Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.