Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.     

Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.     

Effect of environmental haemin upon the physiology and biochemistry of Prevotella intermedia R78

The effect of environmental haemin on the physiology and biochemistry of Prevotella intermedia R78 grown in batch culture was assessed. Extent and rate of growth increased as the environmental haemin concentration was raised. In addition, cell morphology was predominantly cocco-bacillary when cultured in high haemin environments, while bacillary forms were prevalent in low haemin conditions (< 2.5 mumol l-1). Cells harvested from low haemin environments produced greater numbers of extracellular vesicles and greater amounts of peptidolytic activity, haemagglutinating potential and haemin binding activity when compared with cells harvested from high haemin conditions. The results of the present study indicate that aspects of the biochemistry and physiology of P. intermedia are influenced by changes in environmental haemin levels.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.     

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.     

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+-ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore-resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+-ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.0. In the cells grown at pH 7.5, they were moderately observed. The Na+-stimulated ATPase activity of the cells grown at pH 9.5 was about 4-fold higher than that of the cells grown at pH 6.0. Thus, amplification of the Na+-ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Carbon dioxide increases acid resistance in Escherichia coli

AIMS: To investigate how carbon dioxide affects the acid resistance of Escherichia coli. METHODS AND RESULTS: Escherichia coli W3110 was grown in minimal EG medium at pH 7.5, and cells were adapted at pH 5.5 at 37 degrees C with and without supply of carbon dioxide and nitrogen gases. The number of colonies grown on LB medium was measured after cells were challenged in minimal EG medium of pH 2.5 at 37 degrees C under various conditions. When carbon dioxide was supplied at both the acid adaptation and challenge stages, 94% of cells survived after the acid challenge for 1 h, while the survival rates were 50 and 67% when nitrogen gas and glutamate were supplied respectively. After the acid challenge for 3 h, the survival rate observed with the carbon dioxide gas supply was again 2.5-fold higher than those with the nitrogen gas supply. CONCLUSION: Carbon dioxide was shown to participate in the maintenance of high viability under acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information for research into bacterial pathogenesis, fermentation and food preservation.     

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.     

Factors influencing the growth and respiration of rat cerebral astrocytes in primary culture

Cell densities and respiratory rates of astrocytes from neonatal rat brain grown in primary culture were determined after 20–30 days in vitro. Cells grown in flasks reached lower densities (g DNA/cm²) and higher protein: DNA ratios than cells grown in petri dishes. Respiratory rates were lower for cells grown in flasks compared to cells grown in dishes. The pH of the medium in flasks fell below 6.9 between feedings while the pH of the medium in dishes remained at about 7.2. Cells grown in dishes with the medium pH adjusted to 6.8 also showed lower final cell densities, higher protein: DNA ratios, and lower respiratory rates, compared to cells grown under similar conditions at pH 7.5. Intermediate values of each parameter were found in cells grown at pH 7.5 for one week and then at 6.8 for 20 days. We conclude that the effects of ambient pH account for the differences in growth characteristics and respiratory rates of astrocytes grown in dishes versus those grown in flasks.     

Phospholipid-deacylating enzymes of rat stomach mucosa

1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.     

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.     

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.     

Patterns of proteolytic enzyme activities in different tissues of germinating corn (Zea mays L.)

Profiles of pH dependence and activities of live proteolytic enzymes, amino- and carboxypeptidase and endopeptidases active at pH 3.8, 5.4 and 7.5, with casein as substrate, were determined in crude extracts from the various organs of corn seedlings during germination and early development (30°C, dark, 8 d). With respect to the endopeptidases, caseolytic activities at pH 3.8, 5.4 and 7.5 in extracts from endosperm increased concurrently with loss of endosperm N during germination; however, the relative amounts of the pH 7.5 activity were very small. In scutellum extracts, caseolytic activities at both pH 5.4 and 7.5 increased during the initial stages of development but only the increase at pH 5.4 was concurrent with loss of scutellar N. In shoot extracts, caseolytic activities at pH 5.4 and 7.5 were very low and remained relatively constant. There was a progressive increase in shoot N with time. In root extracts, caseolytic activities at pH 5.4 and 7.5 were higher (3-fold) than in shoot extracts. The activity at pH 5.4 remained constant while the activity at pH 7.5 increased during germination. The rate of accumulation of N by the root was low after day 5. The pattern and ratio but not the amounts of the pH 5.4 and 7.5 caseolytic activities of the root were similar to those observed in senescing leaves of field-grown corn. Addition of mercaptoethanol increased (several-fold) the caseolytic activities at pH 3.8 and 5.4, especially the latter, but not the pH 7.5 activity in endosperm extracts and increased the pH 5.4 activity in extracts from scutellum (30%) and roots (30%) while the effect in shoot extracts was negligible. Carboxypeptidase activity was relatively low in young tissue (root tip, 3-d-old shoots) and increased with development of the various organs except the roots (whole) where the activity remained relatively constant. The increases in carboxypeptidase activities were concurrent with decreases in N from endosperm and scutellum; this result indicates that this enzyme in these tissues may be involved (cooperatively with endopeptidases) in the mobilization of reserve protein.Of all the enzymes tested, only carboxypeptidase activity was markedly (in excess of 50%) inhibited by phenylmethylsulfonylfluoride. Only aminopeptidase activity was found in appreciable amounts in endosperm and scutellum of dry kernels. Aminopeptidase activity was highest in organs with high metabolic activity (scutella, shoot, root tips) and decreased in plant parts undergoing rapid loss of nitrogen (endosperm, senescing leaves).Abbreviations AP aminopeptidase - CA caseolytic activity - CP carboxypeptidase - ME mercaptoethanol     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.5-7.5 was also measured. The elevated H+ conductance and lowered H+ flux were shown for cells growing in acidic pH and under anaerobic conditions, when bacteria were fermenting glucose. The atp mutant, which is deprived of the F0F1- adenosine triphosphatase, had less Gm H+ independent of growth conditions. In contrast with wild-type or precursor strain, a remarkable difference in Gm H+ for atp mutant was observed between aerobic and anaerobic conditions; such a difference was significant at pH 4.5. These results could indicate distinguishing pathways determining Gm H+ under anaerobic conditions after the fermentation of glucose at different pH and an input of the F0F1-adenosine triphosphatase in Gm H+. In addition, the effect of osmotic stress was demonstrated with grown cells. Gm H+ and H+ efflux both were increased after hyperosmotic stress at pH 7.5, and these changes were inhibited by N,N\'-dicyclohexylcarbodiimide, whereas these changes were lower in atp mutant. A role of the F0F1-adenosine triphosphatase in osmo-sensitivity of bacteria was confirmed under fermentative conditions.