Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.     

The effect of pH value and temperature on the stability of L-aminoacidoxidase from the venom of the sand viper]

The stability of highly purified L-amino acid oxidase from the sand viper venom remains practically unaffected by the pH-value at 4degreesC between pH 5 and 8, whereas a sharp activity fall was observed on both sides of this range. At temperatures above 30 degreesC the enzyme is stable only at pH 5.0--5.5. The inactivation pH values above 5.5 follows a first-order rate equation with characteristic changes in the absorption and emission spectra of the enzyme. The stability of the enzyme is dependent on the temperature of storage. At pH 7.5 there is a stability minimum at --10 degrees and -- 30 degreesC. At -- 72 degreesC the enzyme is stable practically for an unlimited period of time; temperatures exceeding 50 degrees C rapidly lead to complete inactivation. Also in the cold, the L-amino acid oxidase is most stable at pH 5.5. There are characteristic changes in absorption and emission spectra in the temperature-stability minimum (--15 degreesC) and at temperatures above 30degreesC. The inactivations follow a first-order rate equation. The cold inactivation is reversible. The stability of the enzyme is diminished by some anions and cations at 37 degreesC. The cold inactivation is promoted by several inorganic anions; organic anions and ammonium sulfate prevent cold inactivation.     

A cold-labile acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver. Reactivation and reconstitution of the active species from the inactive monomer

An acetyl-coenzyme-A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold-induced inactivation and dissociation of the enzyme: At a protein concentration of 14 micrograms/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37 degrees C appeared to be optimal for reactivation; warming at 25 degrees C or at 43 degrees C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme-protein concentration of 14 micrograms/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl-CoA (0.5 mM) restored the activity about 50%. High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity. Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4 degrees C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.     

Prevention of cold inactivation of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by NADPH.

Solubilized 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) from rat liver microsomes has been reported to be reversibly inactivated by temperatures below 19 degrees C. Cold inactivation has now been found to be completely prevented by NADPH and by NADP+ at a concentration of 3 mM. NADPH, however, was more active than NADP+ at lower concentrations and prevented 50% of the cold inactivation at 0.2 mM, whereas a 1.1 mM NADPH+ without effect and the substrate 3-hydroxy-3-methylglutaryl coenzyme A prevented only 30% of the cold inactivation at a concentration 50 times greater than the Km value.     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

NADP(+)-malic enzyme from sugarcane leaves: structural properties studied by thermal inactivation

The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.     

Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes

Most C₄ species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C₄ pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0°C) by the divalent cations Mn²⁺, Mg²⁺, and Ca²⁺. There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl₂ (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg²⁺ is required and Mn²⁺ could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg²⁺ had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl₂ was evaluated by incubation at 2 to 17°C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11°C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.     

Production and partial characterization of elastase of Bacillus subtilis isolated from the cervices of human females

Conditions are described for the production of extracellular elastase by Bacillus subtilis. The yield of enzyme was maximum in shake-cultures grown in Syncase medium at 37 degrees C and was stable in culture supernatants. The enzyme, purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration, showed a molecular weight of 25,000 and activity between pH 6.0 and 9.5, with an optimum of 9.0 in Tris-maleate buffer. Elastinolytic activity was maximum in glycine-NaOH buffer and minimum in phosphate buffer. Enzyme activity was adversely affected by temperature greater than or equal to 40 degrees C.     

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.     

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.     

Properties of purified detergent-resistant phospholipase A of Escherichia coli K-12. Inactivation, and protection with detergents and phospholipids.

A crude preparation of membrane-bound phospholipase A (detergent-resistant) in Escherichia coli K-12 cells was found to be quite stable or even apparently activated on incubation at 100 degrees C, but became strikingly thermolabile when it was highly purified and Triton X-100 was removed from the purified enzyme preparation. The rate of inactivation showed a biphasic temperature dependence: inactivation was rapid at 37 degrees C and also above 70 degrees C. Inactivation above 70 degrees C changed the mobility of the enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, but inactivation at 37 degrees C did not affect the electrophoretic mobility. Triton X-100 effectively protected the enzyme against inactivation at 37 degrees C. The concentration required for the protection of the enzyme was more than its critical micelle concentration. Phospholipids, such as phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine, also protected the enzyme against inactivation at 37 degrees C. These results suggest that the binding of hydrophobic compounds stabilizes the enzyme.     

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.     

Characterization of Adenosine deaminase (ADA) in hemolymph of Biomphalaria glabrata.

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 micromols.L(-1). Highest activity was found at 37 degrees C. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 microL of pure hemolymph per assay, pH 6.8, and 37 degrees C. This enzyme showed activities of 834 +/- 67 micromol.min(-1).L(-1) (25 degrees C) and 2029 +/- 74 micromol.min(-1).L(-1) (37 degrees C), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43 degres C or 70% at 50 degrees C for 15 minutes. The ADA lost from 26% to 78% of its activity when hemolymph was pre-incubated at 50 degrees C for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.     

On the partial reactivation of inactivated pantothenase from Pseudomonas fluorescens.

Partial reactivation of inactivated pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) from Pseudomonas fluorescens was studied. After partial inactivation during storing, pantothenase activity is increased by 10-40% when incubated with, for instance, oxalate, oxaloacetate or pyruvate. Reactivation proceedes slowly; with oxaloacetate the stable level of enzyme activity is attained in 20-30 min. The same compounds also cause reactivation of thermally inactivated pantothenase when partial inactivation has occurred at 28-37 degrees C. The amount of the reactivating enzyme form is relatively greater the lower the temperature during inactivation, but it never exceeds 20% of the original amount of active enzyme. Also another, unstable form of pantothenase is formed in thermal inactivation. This form becomes inactivated in a few minutes after the heat treatment, at pH 6-8 and at temperatures between 0 and 10 degrees C. Reactivation causes special problems in enzyme kinetic measurements; for instance, curvature is found in the lines of Ki determination by the Dixon plot.     

Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.     

Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase

The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.     

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 °C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant k _dcalculated for the monomer at 85 °C was 0.029 min^-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 °C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 °C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Particulate carbonic anhydrase in homogenates of human kidney.

About 2% of human kidney carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been found in particulate fractions. Its distribution in the particulate fractions obtained by differential centrifugation suggests that it may be concentrated in the brush border. The particulate enzyme is like red cell carbonic anhydrace C in its susceptibility to inhibition by anions. Particulate carbonic anhydrase is firmly bound to the membrane and is not released by incubation at pH 10.6 and 37 degrees C or by addition of Triton X-100 or deoxycholate. In 10% Triton X-100 at pH 11.3 and 37 degrees C, the particulate enzyme is inactivated with a half time of about 20 min, and this is at least an order of magnitude slower than the inactivation of soluble enzymes in the presence or absence of membranes. The soluble enzymes are inactivated within a few minutes at 25 degrees C in 3-4% sodium dodecyl sulfate, but the particulate enzyme is relatively stable under those conditions, and its half-time of inactivation at 14 degrees C with a detergent-protein ratio of 25 was about 24 h. Gel filtration with Ultragel AcA-44 in sodium dodecyl sulfate indicates that the membrane carbonic anhydrase has a molecular weight of less than 66 000, so its stability is not due to association with large membrane fragments or vesicles. These results suggest that the membrane enzyme may be a different isozyme than the soluble carbonic anhydrases. Although present in relatively small amounts, its localization on the membrane could give it functional significance.     

Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.