Reaction of lysyl oxidase with soluble protein substrates: effect of neutral salts.

Soluble proteins released into the medium of aortic tissues in culture behave as substrates for the enzyme lysyl oxidase. The reaction shows an unusual dependence on the concentration of neutral salts in the assay medium. Practically no enzyme activity was observed in Tris-HCl, 0.005 m, pH 7.6 buffer. However, supplementing the buffer with high concentrations of KCl, KBr, NaCl, and (NH₄)₂SO₄ (in decreasing order of effectiveness) accelerated velocities as much as 10-fold. CaCl₂, KSCN, and KI at increasing concentrations became strongly inhibitory. β-Aminopropionitrile, a specific inhibitor of lysyl oxidase, effectively blocked the catalysis in low and high KCl. The salt-stimulated effects on lysyl oxidase activity were not as noticeable when insoluble proteins were used as substrates. Kinetic studies employing double reciprocal plots revealed that high KCl concentrations (2.0 m) raised the maximum velocity of the reaction but did not alter the apparent K_m. Thus high salt concentrations did not affect the binding of the soluble substrate to the enzyme. In high salts, however, more radioactive substrate proteins appeared to bind to the enzyme, suggesting that the high salt environment increases the fraction of the total enzyme potentially capable of binding to and catalyzing a reaction with the substrate.     

The chemistry of thiamine: Studies on the dimerization of thiazolium salts

In this communication we report on our studies into the previously undetected dimerization chemistry of thiazolium salts. Thiazolium salts with electron-withdrawing substituents, such as 3,4-dimethyl-5-ethoxycarbonylthiazolium iodide, yield acid- and oxygen-sensitive ethylenic dimers under conditions originally used to detect the dimerization of 3-methylbenzothiazolium iodide. The 5-ethoxycarbonyl-4-methyl-3-phenylmethylthiazolium and 5-(2-O-triphenylmethyl-hydroxyethyl)-4-methyl-3-phenylmethylthiazolium bromides yield stable rearranged dimers, rather than the labile ethylenic dimers, under identical conditions. 4-Methyl-5-(2-hydroxyethyl)-3-phenylmethylthiazolium bromide and thiamine hydrochloride yield rearranged dimers which were isolated as their N,O-ketal derivatives when these salts were heated in aprotic solution in the presence of DBN and K₂CO₃, respectively. Rearrangement of the ethylenic dimer of 3-phenylmethylbenzothiazolium bromide to 2-(benzothiazol-2-yl)-2,3-diphenylmethylbenzothiazoline (J. Baldwin, S. E. Branz, and J. A. Walker (1977) J. Org. Chem. 42, 4142) demonstrates that rearranged dimers of these thiazolium salts are produced via a mechanism involving 1,3-sigmatropic rearrangement of intermediate ethylenic dimers. Based on literature precedent we argue that this dimerization chemistry demonstrates the nucleophilic carbene nature of C-2 deprotonated thiazolium salts in aprotic basic solution.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Mechanism of the salt activation of laccase Lac15

Laccases (benzenediol: oxygen oxidoreductases, EC1.10.3.2) can oxidize various substrates, and those which are tolerant to and even activated by salts have attracted a lot of attention due to their application potential in certain industries. The mechanism of the salt activation of laccases is awaiting to be elucidated yet. Our previous study (Li, Xie et al. 2018) supposed that the salt activation of marine laccase Lac15 might be attributed to Cl^- ion specifically binding to some local sites to interfere substrate binding and/or electron transfer. In this study, we found two sites whose mutations resulted in elimination of the salt activation of Lac15’s activity towards catechol and dopamine respectively, and revealed that the mutations affected the activity by altering both E_m and k_cat, demonstrating the supposed mechanism. A model for the salt activation of laccases was accordingly proposed, albeit some details are to be elucidated.     

Salt injury to plants with special reference to cations versus anions and ion activities

Summary In contrast with the toxicities of sulfate and chloride salts added to substrates, the anions SO₄ and C1 were not injurious when accumulated without leaf burning by cotton and tomato plants from atmospheres enriched with SO₂ or HC1 gases. The foregoing results are discussed in terms of cationenzyme interactions which appear to represent at least a major cause of salt toxicity. Although anions are largely unreactive with enzymes it has long been observed that chloride salts in soil solutions are far more toxic than sulfate salts. Five of seven species have shown nearly equal growth repressions on substrates with 100 me/1 of C1 salts versus 200 me of SO₄ salts, each added as 50 per cent Na. The ion activities of the two solutions were equal and the sum of cations in the plant saps were similar. The osmotic differentials (average about 10 atm) between the expressed tissue fluids and these substrate solutions were remarkably uniform within species. It is projected that the downward transport of salts via the phloem provides for root concentrations which supply ions to the xylem and thereby control the uptake of substrate salts.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: I. Characterization of the binding interactions

In this paper we examine molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides. It is shown that the binding affinity (K_oligo) of oligonucleotides of length (l) from two to eight nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding also shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in K_oligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Co-operative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different; here binding is highly salt dependent ($\text{? log Kω}\text{? log}\text{[NaCl]}\text{～- ?7}\text{)}$ and essentially stoichiometric at salt concentrations less than ~0.2 m (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically co-operative (sigmoid) in protein concentration at higher salt concentrations. We demonstrate, by fitting the co-operative binding isotherms to theoretical plots at various salt concentrations and also by measuring binding at very low protein binding density (ν), that the entire salt dependence of Kω is in the intrinsic binding constant (K); the co-operativity parameter (ω) is essentially independent of salt concentration. Furthermore, by determining titration curves in the presence of salts containing a series of different anions and cations, it is shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind two or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.     

The geometry of diphtheria toxoid CRM197 channel assessed by thiazolium salts and nonelectrolytes

The geometry of the channel formed by nontoxic derivative of diphtheria toxin CRM197 in lipid bilayer was determined using the dependence of single-channel conductance upon the hydrodynamic radii of different nonelectrolytes. It was found that the cis entrance of CRM197 channel on the side of membrane to which the toxoid was added at pH 4.8 and the trans entrance on the opposite side at pH 6.0 had effective radii of 3.90 and 3.48 Å, respectively. The 3-alkyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium salts reversibly reduced current via CRM197 channels. The potency of the blockers increased with increasing length of alkyl chain at symmetric pH 6.0 and remained high and stable at pH 4.8 on the cis side. Comparative analysis of CRM197 and amphotericin B pore size with the inhibitory action of thiazolium salts revealed a significant increase in CRM197 pore dimension at pH 6.0. Addition of thiazolium salt with nine carbons alkyl tail increased by ～30% the viability of human carcinoma cells A431 treated with diphtheria toxin.     

The Glutamate Effect on DNA Binding by Pol I DNA Polymerases: Osmotic Stress and the Effective Reversal of Salt Linkage

The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Δn_ions) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Δn_ions in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Δn_ions.Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of ∼ 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to ∼ 150-200 waters released), suggesting that glutamate plays a significant osmotic role.Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.     

Ion-binding and conformation of poly-L-methionine S-methylsulfonium salts in added salt systems

In order to study the type of ion binding and the conformation of several salts of poly-L -methionine S-methylsulfonium hydroxide, the viscosity, conductance, counterion activity, and optical rotatory dispersion of the polysalts were measured in systems with a small amount of added salt. It was shown that the ion binding of chloride and bromide salts was of a diffuse binding type due only to the electrostatic potential of the polyion, and that both polysalts underwent no conformational transition by the addition of a simple salt, NaCl for chloride salt and NaBr for bromide salt, and retained a random coil conformation. Iodide and thiocyanate salts showed a conformational change, probably from the random coil into the β form, with increasing concentrations of NaI and NaSCN, respectively. On the other hand, perchlorate salt existed in the α-helix conformation in part even in pure aqueous solution, and the fraction of α-helix increased on the addition of NaClO₄. On considering several possible situations, it is suggested that there is a specific and strong interaction between the polyion and the small counterion in iodide, thiocyanate, and perchlorate salts.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Tissue-Specific and Cation/Anion-Specific DNA Methylation Variations Occurred in C. virgata in Response to Salinity Stress

Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na₂SO₄, NaHCO₃, Na₂CO₃) and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na₂CO₃> NaHCO₃> Na₂SO₄> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Changes in the catalytic properties and substrate specificity of Bacillus sp. US149 maltogenic amylase by mutagenesis of residue 46

Maltogenic amylase from Bacillus sp. US149 (MAUS149) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of residue Asp46 in the specificity and catalytic properties of MAUS149 by using site-directed mutagenesis. Three mutated enzymes (D46V, D46G and D46N) were constructed and studied. The three mutants were found to be similar to the wild-type MAUS149 regarding thermoactivity, thermostability and pH profile. Nevertheless, the kinetic parameters for all the substrates of the mutant enzymes D46V and D46G were altered enormously as compared with those of the wild type. Indeed, the K _m values of MAUS149/D46G for all substrates were strongly increased. Nevertheless, the affinity and catalytic efficiency of MAUS149/D46V toward β-CD were increased fivefold as compared with those of MAUS149. Molecular modelling suggests that residue D46 forms a salt bridge with residue K282. This bond would maintain the arrangement of side chains of residues Y45 and W47 in a particular orientation that promotes access to the catalytic site and maintains the substrate therein. Hence, any replacement with uncharged amino acids influenced the flexibility of the gate wall at the substrate binding cleft resulting in changes in substrate selectivity.     

Correlation Between Thermal Aggregation and Stability of Lysozyme with Salts Described by Molar Surface Tension Increment: An Exceptional Propensity of Ammonium Salts as Aggregation Suppressor

Protein aggregation is a critical problem for biotechnology and pharmaceutical industries. Despite the fact that soluble proteins have been used for many applications, our understanding of the effect of the solution chemistry on protein aggregation still remains to be elucidated. This paper investigates the process of thermal aggregation of lysozyme in the presence of various types of salts. The simple law was found; the aggregation rate of lysozyme increased with increasing melting temperature of the protein (T _m) governed by chemical characteristics of additional salts. Ammonium salts were, however, ruled out; the aggregation rates of lysozyme in the presence of the ammonium salts were smaller than the ones estimated from T _m. Comparing with sodium salts, ammonium salts increased the solubility of the hydrophobic amino acids, indicating that ammonium salts adsorb the hydrophobic region of proteins, which leads to the decrease in aggregation more effectively than sodium salts. The positive relation between aggregation rate and T _m was described by another factor such as the surface tension of salt solutions. Fourier transform infrared spectral analysis showed that the thermal aggregates were likely to form β-sheet in solutions that give high molar surface tension increment. These results suggest that protein aggregation is attributed to the surface free energy of the solution.     

Production of aflatoxin by Aspergillus parasiticus NRRL-2999 during growth in the presence of curing salts

Aspergillus parasiticus NRRL-2999 was inoculated into meat mixtures with curing salts and into yeast extractsucrose (YES) and sucrose-ammonium salts (SAS) broth with and without curing salts to determine if the presence of curing salts significantly affected growth and aflatoxin production by the mold. The effect of individual curing salts or curing salt mixtures on growth and toxin elaboration by the aspergillus was substrate dependent. When YES broth contained 100 ppm of NaNO₂, 2% NaCl, or 1 or 2% NaCl plus 200 ppm of NaNO₂ or 200 ppm of NaNO₃, growth and/or aflatoxin production was depressed. Biosynthesis of aflatoxin B₁ was enhanced by presence of 1 and 4% NaCl in YES broth. The SAS broth containing only NaCl or NaCl combined with nitrite or nitrate yielded less aflatoxin than did control broth or no aflatoxin at all. When compared to the control, an increase in growth and amount of aflatoxin occurred in SAS broth which contained 200 ppm of NaNO₃. Sausages containing 100 and 200 ppm NaNO₂ and no NaCl supported more mold growth and aflatoxin production than did control sausage with 3 % NaCl and 100 ppm of NaNO₂. Addition of 2 and 3 % NaCl and no nitrite to sausage resulted in less aflatoxin than in control sausage.     

Molecular ruler of tripeptidylpeptidase II: mechanistic principle of exopeptidase selectivity

The structure of tripeptidylpeptidase II (TPPII) has shown that it belongs to the group of exopeptidases which use a double-Glu motif to convey aminopeptidase activity. TPPII has been implicated in vital biological processes. At least one of these, antigen processing, requires the involvement of its endopeptidase activity. In order to understand the extent and molecular basis of this unusual functional promiscuity we have performed a systematic kinetic analysis of wild type Drosophila melanogaster TPPII and five point mutants of the double-Glu-motif (E312/E343) involving natural substrates. Unlike the known double-Glu motives of other exopeptidases, the double-Glu motif of TPPII is distinctly asymmetrical: E312 is the crucial determinant of the aminotripeptidolytic ruler mechanism. It both blocks the active-site cleft at substrate position P4 and forms a salt bridge with the N-terminus of the substrate. In contrast, E343 forms a much weaker salt bridge than E312 and it does not have a blocking role. An endopeptidase substrate can bind at relatively high affinity if the length of the substrate permits binding to several S′ sites. However, the lacking alignment of the substrate by the double-Glu motif causes the endopeptidolytic K_cat/K_M of TPPII to be very low.     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.