Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-[LDH-like] malate dehydrogenase from the halophilic archaeon Haloarcula marismortui

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric. To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH). Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters. In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted. Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl. At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration. The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized. The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs. A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot. The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH.     

Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 A resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui

Previous biophysical studies of tetrameric malate dehydrogenase from the halophilic archaeon Haloarcula marismortui (Hm MalDH) have revealed the importance of protein-solvent interactions for its adaptation to molar salt conditions that strongly affect protein solubility, stability, and activity, in general. The structures of the E267R stability mutant of apo (-NADH) Hm MalDH determined to 2.6 A resolution and of apo (-NADH) wild type Hm MalDH determined to 2.9 A resolution, presented here, highlight a variety of novel protein-solvent features involved in halophilic adaptation. The tetramer appears to be stabilized by ordered water molecule networks and intersubunit complex salt bridges "locked" in by bound solvent chloride and sodium ions. The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions. The analysis of the crystal structures showed that halophilic adaptation is not aimed uniquely at "protecting" the enzyme from the extreme salt conditions, as may have been expected, but, on the contrary, consists of mechanisms that harness the high ionic concentration in the environment.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

Effects of inorganic salts on tissue permeability

Inorganic solutes are shown to alter the permeability of root and leaf tissues. Experiments with beet root tissues reveal that CaCl(2) decreases leakage of betacyanin from the tissue, that (NH(4))(2)SO(4) increases leakage, and that each salt can relieve the effects of the other. A comparison of cations and anions shows a range of effects with the various solutes. Experiments with Rumex obtusifolius L. leaf discs reveal that whereas CaCl(2) defers the development of senescence, (NH(4))(2)SO(4) hastens senescence and increases the leakage of materials out of the leaf discs. The solute effect on Rumex obtusifolius L. is prevented by gibberellin. CaCl(2) can relieve the (NH(4))(2)SO(4) effect. The results are interpreted as indicating that the inorganic solutes may serve to alter the permeability of membranes through alterations of interactions between water and macromolecules in the tissues; the interpretation is consistent with the evidence for opposite effects of Ca and NH(4), the effective concentrations being about 10(-3)m, and the reversibility of the effects of one solute by another of opposite stabilization-destabilization effect.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Pendrin in the mouse kidney is primarily regulated by Cl- excretion but also by systemic metabolic acidosis

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

Influence of protein surface morphology on the ultrafiltration flux resistance of bovine serum albumin.

The effect of added ethanol and (NH(4))(2)SO(4) on the flux decline index (FDI) of bovine serum albumin (BSA) and a fatty acid-poor derivative (BSA/FAP) was examined. Ternary phase diagrams of the two protein species indicated that the concentration polarization (CP) layer on the surface of a nonadsorbing 10 000 MWCO regenerated cellulose membrane had principally a packed bed structure up to 33 wt % ethanol and 21 wt % (NH(4))(2)SO(4). Intrinsic viscosity and turbidity analysis were conducted to determine the degree of intra- and interprotein interactions within this packed bed morphology. With BSA/FAP, the effects of these two interactions tended to counterbalance each other, so the FDI of this protein was not strongly influenced by solute addition. In contrast, the adsorption of fatty acids to BSA caused the protein to expand, producing a less rigid CP layer with a higher FDI. However, the addition of ethanol led to protein compression, reducing this effect. The presence of fatty acids also produced a more associated BSA in salt solution, which increased flux resistance. The results obtained for both proteins indicate that an FDI minimum is observed when a noninteraction hard sphere structure is present in the CP layer.     

Hydrophobic interaction adsorption of hen egg white proteins albumin, conalbumin, and lysozyme

Hydrophobic adsorption equilibrium data of the hen egg white proteins albumin, conalbumin, and lysozyme were obtained in batch systems, at 25 degrees C, using the Streamline Phenyl resin as adsorbent. The influence of three types of salt, NaCl, Na(2)SO(4), or (NH(4))(2)SO(4), and their concentration on the equilibrium data were evaluated. The salt Na(2)SO(4) showed the higher interaction with the studied proteins, thus favoring the adsorption of proteins by the adsorbent, even though each type of salt interacted in a distinct manner with each protein. The isotherm models of Langmuir, Langmuir exponential, and Chen and Sun were well fitted to the equilibrium data, with no significant difference being observed at the 5% level of significance. The mass transfer model applied simulated correctly adsorption kinetics of the proteins under the studied conditions.     

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.16 M (NH4)2SO4 and was maximal at 0.6-1.0 M. The maximal activation of the enzyme was observed within 3 min of pretreatment and was not readily reversible. The effect was specific for the NH+4 ion since pretreatment of membranes with other NH+4 salts could activate the enzyme, whereas pretreatment with NaCl or KCl could not. Pretreatment of plasma membranes with (NH4)2SO4 eliminated the sensitivity of the enzyme to the inhibitory effect of guanine nucleotides. (NH4)2SO4 pretreatment also significantly attenuated the inhibition by guanine nucleotides of cAMP binding to its plasma membrane receptor. The effect of (NH4)2SO4 on GTP inhibition of cAMP binding to its receptor was even more dramatic when the salt was present in the binding assay. (NH4)2SO4 also increased the ADP-ribosylation by cholera toxin of a 39,000-Da membrane protein. The data support the hypothesis that (NH4)2SO4-induced changes in adenylate cyclase and the cAMP receptor are due to an alteration of a putative G protein.     

Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by Methylosinus trichosporium OB3b and Maine Forest Soils

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of Methylosinus trichosporium OB3b. The comparable level of inhibition by equinormal concentrations of NH(4)Cl and (NH(4))(2)SO(4) and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH(4)Cl than (NH(4))(2)SO(4) in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

Salt-induced structural changes in nucleosomes.

Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococcal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes was examined in the presence and absence of 10 mM MgCl2. Nucleosomes and oligonucleosomes are insoluble over a broad range of salt concentration. Nucleosomes are insoluble in larger than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 allowing analyses of changes in nucleosomal DNA by C.D. spectroscopy. Nucleosomes are insoluble in less than or equal to 120 mM (NH4)2SO4 containing 10 mM MgCl2 as demonstrated by turbidity measurements. We conclude that the insolubility of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.     

Substrate induction and statistical optimization for the production of chitosanase from Microbacterium sp. OU01

The chitosanase production was markedly enhanced by substrate induction, statistical optimization of medium composition and culture conditions by Microbacterium sp. OU01 in shake-flask. A significant influence of (NH(4))(2)SO(4), MgSO(4).7H(2)O and initial pH on chitosanase production was noted with Plackett-Burman design. It was then revealed with the method of steepest ascent and response surface methodology (RSM) that 19.0g/L (NH(4))(2)SO(4), 1.3g/L MgSO(4) and an initial pH of 2.0 were optimum for the production of chitosanase; colloidal chitosan appeared to be the best inducer for chitosanase production by Microbacterium sp. OU01. This optimization strategy led to the enhancement of chitosanase from 3.6U/mL to 118U/mL.     

Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH(4))(2)SO(4). Retention on these columns was greatest at the highest concentrations of (NH(4))(2)SO(4). By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH(4))(2)SO(4)-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine-Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.     

In vitro penetration of zona pellucida of salt-stored bovine oocytes before and after maturation by frozen-thawed spermatozoa

Bovine oocytes, before and after maturation in culture, were stored in PBS with 2 M-(NH(4))(2)SO(4) + 0.1% dextran or 2 M-(NH(4))(2)SO(4) + 40 mM-Hepes + 0.5% dextran and were inseminated with frozen-thawed spermatozoa in BO medium with caffeine (5 mM) and heparin (10 mug/ml). The penetration rates of mature oocytes were very low (19 to 24%) and not significantly different between the two salt solutions in which the oocytes were stored for 2 to 89 days. Significantly lower (P < 0.01) penetration rates were observed in immature (7 to 8%) than in mature (20 to 21%) oocytes stored in the two solutions. The synergistic effect of caffeine and heparin was observed in the penetration rate of fresh mature oocytes but not in the stored oocytes, indicating the difficulty of assessing sperm capacitation and/or acrosome reaction of salt-stored mature bovine oocytes under the present condition. Using 0.1% protease the solubility of the zonae decreased in salt-stored but not in fresh oocytes, but there was no significant difference between the immature and mature oocytes regardless of storage in the salt solutions. It appears from these results that some alteration was induced in the nature of zona glycoprotein by ammonium sulfate solution.     

The purification of cholinesterase from horse serum

A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85-90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.