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.     

Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.     

怀头鲇早期发育阶段消化酶及碱性磷酸酶活性变化(英文)

采用动物性饵料和人工饲料培育1～10日龄怀头鲇(Silurus soldatovi)仔稚鱼,分析测定了全鱼酸性、碱性蛋白酶、淀粉酶、脂肪酶以及碱性磷酸酶的活性。结果表明:孵化后3天开口期仔鱼已具有较高的碱性蛋白酶活性,5日龄时碱性蛋白酶比活力达到较高值,8日龄时出现低值,总体变化呈波动上升趋势;酸性蛋白酶活性在1～8日龄处于较低水平,8日龄后开始迅速升高;淀粉酶活性在5日龄左右达到最高值,随后酶活性开始下降至较低水平;脂肪酶活性变化波动较大,表现为双峰型,两个峰值分别出现在3～4日龄和6～8日龄。摄食动物性饵料仔稚鱼消化酶活性和碱性磷酸酶活性均高于摄食人工饲料。在整个早期发育过程中,碱性蛋白酶比酸性蛋白酶活性高,碱性蛋白酶、淀粉酶比活力在约8日龄仔稚鱼转变期明显下降,而酸性蛋白酶活性开始迅速升高,这说明消化酶活性的变化与仔稚鱼发育过程中消化机能转换具有相关性。怀头鲇在10日龄内碱性磷酸酶活性呈上升趋势,表明怀头鲇胃肠道功能的逐步发育完善。     

Comparison of kinetic and optical changes during guanidine denaturation of intestinal alkaline phosphatase

The modifications of the activity of calf intestinal Alkaline Phosphatase treated with moderate amounts of guanidinium chloride are compared with the conformational changes observed by ultraviolet absorbance and intrinsic fluorescence. The time course of catalytic and optical properties of the treated enzyme develops through two distinct steps: an instantaneous and a time-dependent one. The immediate effect of guanidine is to lower emission yield, to shift the emission maximum of the enzyme to longer wavelengths and to enhance the absorbance of the protein. The rapid conformational transition determines a paradoxical activation at low effector concentration (below 0.88 M) and an inhibition at higher amounts. The following marked decay of enzyme activity with time is related to spectroscopically detectable changes. Temperature influences both kinetic and structural aspects of the process and facilitates guanidine action.     

Effects of various anions on the Raman spectrum of halorhodopsin.

Resonance Raman experiments were conducted to probe and understand the effect of various anions on halorhodopsin. These included monoatomic anions Cl- and Br-, which bind to the so-called halorhodopsin binding sites I and II, and polyatomic anions NO3- and ClO4-, which bind to site I only. The two types of ions clearly show different effects on the vibrational spectrum of the chromophore. The differences are not localized to the Schiff base region of the molecule, but extend to the chromophore structure-sensitive fingerprint region as well. We find that the protonated Schiff base frequency is at 1,633 cm-1 for Cl- and Br- ions, as reported previously for Cl-. However, we find that two Schiff base frequencies characterize halorhodopsin upon binding of the polyatomic anions. One frequency lies at the same location as that found for the monoatomic anions and the other is at 1,645 cm-1. Halorhodopsin with bound NO3- and ClO4- thus may consist of two heterogeneous structures in equilibrium. This heterogeneity does not seem to correlate with a retinal isomeric heterogeneity, which we can also demonstrate in these samples. The results suggest that anions binding to site I do not bind to the Schiff base directly, but can influence chromophore and/or protein conformational states.     

The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase.

In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine. The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site. However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme. The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site. However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl. 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme. The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes. These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group.     

Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium.     

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.     

Effects of immobilization on electromyogram power spectrum changes during fatigue.

The maximal force and median frequency (MF) of the electromyogram (EMG) power density spectrum (PDS) have been compared in disused (6 weeks' immobilization) and control (contralateral) human adductor pollicis muscles during fatigue induced by voluntary or electrically-triggered (30 Hz) contractions. The results indicated that after 6 weeks' immobilization, MF was not significantly different in disused and control muscles although the force and integrated EMG were drastically reduced during a maximal voluntary contraction (MVC; by 55% and 45%, respectively, n = 8). During sustained 60 s MVC, the force decreased at the same rate in immobilized and control muscles, but the shift of MF towards lower frequency values was smaller (P less than 0.05) in disused muscle as compared to control by (14% vs 28%, respectively). In electrically-induced fatigue, the force decrease and the MF shift were larger after inactivity (41% and 43% in one subject, and 50% and 54% in the other subject, respectively) as compared to control (29% and 34% in one subject, and 37% and 38% in the other subject, respectively). These results emphasize the caution that should be exercised when EMG signals are quantified by computing the power density spectrum. The different effects of fatigue during voluntary and electrically-imposed contractions in disused and control muscles indicated that immobilization induced changes in the neural command for the contraction which compensated, at least in part, for its decreased contractile efficiency and resistance to fatigue.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Rye germ acid phosphatase: properties of the enzyme and its activation by lectins

Acid phosphatase (EC 3.1.3.2) from rye germs is a glycoprotein of M, 90000 with subunit structure. The pH optimum for pNPP hydrolysis is 5.4. The best substrates for the enzyme are pNPP, PP_i and ATP. In the presence of plant lectins an increase in AcPase activity was found. ConA causes a 20% decrease of K_m^app and a 50% increase of V_max^app with pNPP as substrate.     

Experimental evidence for interactions between bacterial peptidase and alkaline phosphatase activity in the Baltic Sea

From the observed pattern of aminopeptidase and alkaline phosphatase activities in the Baltic Sea, the question arose whether there is an interaction between the activities of both enzymes. In experiments with 0.8 m filtered seawater, the effects of commercial alkaline phosphatase on bacterial aminopeptidase, the effects of commercial peptidase on bacterial alkaline phosphatase activity (APA), and the effects of proteins, carbohydrates and inorganic nutrients on the activities of both enzymes were investigated.Addition of commercial alkaline phosphatase stimulated bacterial aminopeptidase activity and, similarly, the addition of commercial peptidase increased the APA in bacteria. The proteins, albumin and casein, stimulated aminopeptidase activity and APA simultaneously. Experiments using ammonium and glucose suggested that stimulation of APA by peptidase could be mediated by nitrogen and carbon availability. There were also some indications that stimulation of aminopeptidase activity by alkaline phosphatase functioned by catalysing phosphate release from organic phosphorus compounds.     

Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye: DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Influence of anions on the activation of carbamoyl phosphate synthetase (ammonia) by acetylglutamate: implications for the activation of the enzyme in the mitochondria

Rat liver carbamoyl phosphate synthetase is shown to be inhibited by anions competitively with acetylglutamate (the allosteric activator of the enzyme) with a potency decreasing in the order NO3- greater than SO4(2-) greater than Cl- approximately HCO3-. Inhibition by chloride accounts for most of the inhibition reported [Lund, P., and Wiggins, D. (1987) Biochem. J. 243, 273-276] in Tris buffer. Mes, acetate, and isethionate give little or no inhibition and phosphate inhibits noncompetitively. Plots of the KA value for acetylglutamate versus the concentration of chloride or nitrate are curved upward and binding assays demonstrate that the inhibitory anions displace acetylglutamate from the enzyme. Thus, the anions may compete with the carboxyls of acetylglutamate for positive charges at the binding site. Of the organic anions found in the mitochondrial matrix, alpha-ketoglutarate, malate, succinate, and citrate increase substantially the KA for acetylglutamate. Changes in the concentrations of ATP, HCO3-, NH4+, and Mg2+, and high concentrations of protein (60 mg/ml serum albumin) influence the KA value. Changes in the concentration of the enzyme have no effect. Under assay conditions approaching the ionic, buffer, and substrate concentrations expected to occur in the mitochondrial matrix, the KA value for acetylglutamate is 27 microM and the Vmax is decreased about 50%. These results indicate that physiological changes in the level of acetylglutamate significantly influence the degree of activation of carbamoyl phosphate synthetase in vivo.