Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase by pyridoxal-5'-phosphate.

Camel lens zeta-crystallin was inhibited by pyridoxal-5'-phosphate (PAL-P) and o-phthalaldehyde. PAL-P inactivated zeta-crystallin in a time- and concentration-dependent manner. The initial rate of inactivation followed pseudo-first-order kinetics with the second-order rate constant of 91 M-1 s-1. The modified enzyme showed the characteristic absorption peak at 325 nm indicative of the formation of phosphopyridoxallysine. Quantitative analysis suggested the incorporation of 1 mole of PAL-P/subunit of enzyme. NADPH was able to substantially protect zeta-crystallin against PAL-P inactivation, whereas the substrate 9,10-phenanthrenequinone (PQ) did not provide any protection. Inhibition of zeta-crystallin by PAL-P was uncompetitive with NADPH (Ki=37 microM) and non-competitive with respect to the substrate (Ki=57 microM). Inhibition of zeta-crystallin by o-phthalaldehyde was used to establish the location of an essential lysine residue. Incubation of zeta-crystallin with o-phthalaldehyde resulted in the formation of an isoindole derivative that had a characteristic fluorescence spectrum. This suggested that a lysine residue is located within 3 A of a cysteine residue at the NADPH binding region. SDS-PAGE showed the o-phthalaldehyde-modified enzyme remained largely monomer (approx. 80%), although bands corresponding to dimer and tetramer forms were also present. These results suggested that an essential lysine residue is located in the vicinity of the NADPH binding site. This residue may simply ensure the proper binding of NADPH to the active site of zeta-crystallin.

Hydrophobicity of the NADPH binding domain of camel lens zeta-crystallin.

Interaction of camel lens zeta-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-zeta-crystallin binding. Quantitative analysis indicated that 1 mole zeta-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta-crystallin, possibly at the NADPH binding site. alpha-Crystallin as well as NADPH protected zeta-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K(i) of 2.3 microM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 microM for the zeta-crystallin-NADPH-ANS complex. The K(i) being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.

High-affinity binding of NADPH to camel lens zeta-crystallin.

Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.

Kinetic properties of camel lens zeta-crystallin.

Camel lens zeta-crystallin is an NADPH:quinone oxidoreductase, showing limited quinone substrate specificity: among the quinones tested, the orthoquinones were the best substrates. The kinetic mechanism of NADPH:quinone oxidoreductase activity of zeta-crystallin was investigated by steady-state initial velocity and product-inhibition studies. The results were consistent with the enzyme having a ping-pong mechanism and the intrinsic K(m) values for NADPH and 9,10-phenanthrenequinone (PQ) were estimated to be 48.0 +/- 2.5 and 36.0 +/- 1.5 microM, respectively. NADP+ showed a mixed type of inhibition with respect to NADPH, while it was a competitive inhibitor with respect to PQ. Dithiothreitol (DTT) was a strong competitive inhibitor with respect to PQ with a Ki value of 50 microM. Similarly, 2, 3-dimercaptopropanol inhibited the enzyme activity with a Ki value of 260 microM. The enzyme was inactivated by sulfhydryl groups, modifying agents such as 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) and N-ethyl-maleimide. The substrate specificities and kinetic properties of camel lens NADPH:quinone oxidoreductase reported here distinguish it from previously characterized quinone oxidoreductases. This paper reports, for the first time, on the kinetic mechanism of camel lens zeta-crystallin.

Kinetic properties of purified carnitine acetyltransferase from the skeletal muscle of Arabian camel (Camelus dromedarius).

The kinetic properties of carnitine acetyltransferase from the skeletal muscle of the Arabian camel (Camelus dromedarius) were studied. The enzyme showed an optimum pH between 7.2 and 8.2. Reciprocal plots of data obtained by varying one substrate concentration while keeping the other constant revealed lines that converged on the abscissa, indicating that the enzyme possible follows a random mechanism of catalysis. The Kms for L-carnitine and acetyl-coenzyme A were 244 and 44 microM respectively, while those for acetyl-DL-carnitine and coenzyme A (Co A) were 307 and 39 microM respectively. The Km for one substrate was found to be independent of the concentration of the second substrate used. Corresponding Vmax values for L-CA, acetyl-Co A, acetyl-DL-carnitine and Co A are 98, 98, 102 and 100 mumol min-1 mg-1 protein respectively. The low Km obtained for acetyl-DL-carnitine suggests an adaptive mechanism in this desert species for enduring prolonged dry spells without food and water.

Purification and characterization of acetylcholinesterase from desert cobra (Walterinnesia aegyptia) venom.

Acetylcholinesterase (AChE) has been identified and purified from the venom of desert cobra (W aegyptia) to apparent homogeneity using a TSK G 3000 SW gel filtration column and a Mono Q anion-exchange column. AChE was purified to homogeneity as established by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The specific activity of AChE was 357 IU/mg with acetylthiocholine iodide as substrate. The denatured W aegyptia venom AChE displayed a molecular mass of 67000 +/- 3000 Da suggesting it was a single polypeptide. Isoelectric focusing of AChE revealed that the enzyme exists in different isoforms, with isoelectric points ranging between pH 7.4-7.9. The kinetic parameters (Km and Vmax) and IC50 of AChE inhibition by procaine, tetracaine and physostigmine were investigated in the present study.

Inhibition of human acetylcholinesterase by cyclophosphamide.

The inhibitory effect of cyclophosphamide (CP) on human erythrocyte membrane bound acetylcholinesterase (AChE) was investigated in the present study. It was found that CP inhibits the AChE reversibly with an IC50 of 511 microM. The Michaelis-Menten constant (Km) was 132 microM for AChE in the control system; a value increased by 78% in the CP treated system. The Vmax was 73.8 mumol/h/mg protein for the control system. The Lineweaver-Burk plot and Dixon plot indicated that the nature of this inhibition is of the linear mixed type, i.e., partially competitive and purely noncompetitive. The values of Ki and KI were estimated as 378 and 582 microM, respectively. The KI was greater than Ki indicating that noncompetitive inhibition was predominant over competitive.

Purification and some properties of camel lens crystallins.

Five major crystallin fractions were found in camel lens fractionated by Sepharose CL-6B column chromatography. These crystallin fractions were named alpha high (alpha H), alpha low (alpha L), beta high (beta H), beta low (beta L) and gamma (gamma) by comparison with the elution profiles and molecular weights of rabbit crystallins. The amino acid composition and isoelectric focusing bands for crystallins of camel and rabbit were remarkably similar, but individual differences were found. By means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two alpha H crystallin subunits, of 23K and 20K molecular weight, were found in camel and rabbit. Likewise, a single 21K molecular weight band was found in the gamma crystallin of camel and rabbit. In camel, the beta high crystallin consisted of five major subunits, while rabbit beta high crystallin consisted of only three subunits. On SDS-PAGE, camel and rabbit beta low crystallins both showed two major subunits of 27K and 23K molecular weight but camel beta low crystallin showed an additional 35K molecular weight subunit. Characterization of camel lens crystallins may contribute to understanding the effect of aging on aggregation of camel crystallins.

Determination of L-carnitine, acylcarnitine and total carnitine levels in plasma and tissues of camel (Camelus dromedarius).

The total, free and acyl carnitine levels were measured in plasma and tissues of the Arabian camel (Camelus dromedarius). Significant variation in carnitine concentrations were observed in plasma and tissues of the camel when compared with other animal species. A higher proportion of acyl carnitine was found in plasma and skeletal muscle of the camel than other animal species. Among the camel tissues, skeletal muscle possessed the highest amount of carnitine while the lowest amount was found in kidney. The higher carnitine content and a higher proportion of acyl carnitine in plasma and tissues of the Arabian camel suggest an adaptive mechanism that could be common to desert animal species.

Glycation of human lens proteins from diabetic and (nondiabetic) senile cataract patients.

Glycation (nonenzymatic glycosylation) in the human lens (cortex and nucleus) in senile (nondiabetic) and diabetic cataracts was studied by measuring the extent of early and late glycation products, the content of free epsilon-amino groups and the formation of disulfide bonds in the soluble lens proteins. There was a significant (p < 0.001) increase in early and late glycation in the lens nucleus compared to the cortex in both the senile and diabetic groups. Overall these changes were much larger in the diabetic group. The concentration of free epsilon-amino groups was decreased in the senile nucleus as well as in the diabetic nucleus when compared with the senile and diabetic cortex (p < 0.001). Disulfide bond content was in the order of diabetic nucleus > diabetic cortex > senile nucleus > senile cortex. Glycation of the lens proteins is a generalized feature which is enhanced in the diabetic lens compared to senile lens proteins and is associated with a decrease in free epsilon-amino groups and an increase in disulfide bonds formation in the lens proteins.

Characterization of zeta-crystallin inhibition by juglone.

Guinea pig lens zeta-crystallin showed hyperbolic saturation curves with 9,10-phenanthrenequinone (PAQ). 5-hydroxy-1,4-naphthoquinone (juglone) and NADPH. Whereas camel lens zeta-crystallin showed hyperbolic saturation curves only with PAQ and NADPH, but slightly segmoidal with juglone. For both enzymes PAQ was the preferred substrate. The catalytic center activity (Kcat) values indicated that camel zeta-crystallin catalyzed the reduction of PAQ more efficiently than the guinea pig lens zeta-crystallin, although the Km values of the two enzymes for this quinone were very similar. The guinea pig lens zeta-crystallin catalyzed the reduction of Juglone far more efficiently than that of the camel lens zeta-crystallin. Juglone did not serve as an efficient substrate for both zeta-crystallins compared to PAQ and appeared to act as a potent competitive inhibitor, with Kl values of 75 nM and 20 microM for guinea pig lens zeta-crystallin and camel lens zeta-crystallin, respectively. Thus, the camel lens zeta-crystallin was less active toward juglone as a substrate as well as less sensitive to its inhibitory action, when compared with guinea pig lens zeta-crystallin. The inhibition mechanism of guinea pig and camel lens zeta-crystallin by juglone is discussed.

Inhibition of zeta-crystallin by Coumarins: a structure-activity study.

A structure-activity study was carried out to determine the important groups of coumarin derivatives in inhibiting the oxidoreductase activity of the camel lens zeta-crystallin. Coumarin, 4-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, dicoumarol, and warfarin were screened for their inhibitory effect on zeta-crystallin activity. The sequence of potency for the inhibitors was dicoumarol > 4-hydroxycoumarin > warfarin > > coumarin. 7-Hydroxy-4-methylcoumarin was ineffective as an inhibitor. Only dicoumarol, 4-hydroxycoumarin, and warfarin were found to inhibit the oxidoreductase activity in micromolar ranges. All tested inhibitors seem to act in reversible and time-independent manner. Concentration causing 50% inhibition of the enzyme activity (IC50 value) was 34 microM for dicoumarol, 76 microM for 4-hydroxycoumarin, and approximately 515 microM for warfarin, while 1 mM coumarin showed less than 10% inhibition. Kinetic analysis revealed inhibition of camel lens zeta-crystallin by coumarin derivatives to occur in a competitive manner with respect to dichlorophenolindophenol (DCIP) as an electron acceptor and uncompetitive manner with respect to NADPH as an electron donor. The Ki values were found to be 16 microM for dicoumarol, 40 microM for 4-hydroxycoumarin, and 220 microM for warfarin. The structure-activity relationship of coumarin derivatives indicates that the phenolic hydroxyl group at the C-4 position in the coumarin skeleton is important for the maximal inhibition.

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase activity by Cibacron blue.

Camel lens zeta-crystallin/NADH:quinone oxidoreductase activity was inhibited by Cibacron blue 3GA (CB) with 9.10-phenanthrenequinone (PQ) as an electron acceptor and NADPH as an electron donor in a time-independent and concentration dependent manner. The IC50 value of CB was 50 nM. The Lineweaver-Burk plots and the secondary plots indicated that the inhibition was linear mixed type (partial competitive and pure noncompetitive) with respect to NADPH and noncompetitive with respect to PQ. The estimated inhibition constant (Ki) values were 26.0 nM for NADPH and 55.0 nM for PQ respectively, suggesting that CB has high affinity towards the NADPH binding site. The secondary plots of inhibition with respect to NADPH, also indicate a dissociation constant (Ki) value of 68.0 nM for the zeta-crystallin-NADPH-CB complex. This Ki being greater than the Ki value suggests that noncompetitive inhibition is predominant over competitive inhibition at the NADPH binding site.

Camel lens zeta-crystallin kinetics and its inhibition by dicoumarol.

zeta-Crystallin a novel NADPH quinone oxidoreductase of the camel lens is capable of reducing a non quinone compound such as 2,6-dichlorophenolindophenol (DCIP) which is mediated by NADPH. A classical Michaelis-Menten kinetics were exhibited for both DCIP and NADPH with Km values of 15.3 microM and 7.0 microM respectively, at pH 7.8. The Vmax was 1.60 mumol./min.mg protein. The results of steady-state kinetic analysis indicated that the reaction proceeds through a Ping-Pong mechanism. Dicoumarol was found to be a competitive inhibitor of zeta-crystallin with respect to DCIP with a Ki value of 14.6 microM and showed uncompetitive inhibition with respect to NADPH with a Ki value of 36.2 microM.

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus.

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.

Isolation of camel brain actin--comparison of its biochemical properties with those of camel skeletal muscle, heart muscle and rabbit skeletal muscle actins.

1. Actins were purified from camel brain, skeletal muscle and heart muscle and their properties were compared. 2. Individual actins were homogeneous and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 3. Isoelectric focusing analysis of camel skeletal muscle and heart muscle actin showed a single polypeptide of the alpha-species, while camel brain actin showed two polypeptides of the beta- and gamma-species typical of non-muscle actin. 4. Actins from camel skeletal muscle and heart muscle showed a greater degree of similarity to each other and to rabbit skeletal muscle actin and showed some differences from camel brain actin, as confirmed by amino acid analysis and one-dimensional peptide mapping.

Involvement of a disulfide bridge in catalytic activity of camel lens xi-crystallin.

Chemical modification studies were performed to elucidate the role of cysteine residues in the catalytic activity of camel lens xi-crystallin. 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the camel lens xi-crystallin revealed that it contained 3 SH-groups and a disulfide bridge per subunit. One of the three SH-groups was readily accessible, whereas the other two were buried. Inactivation of xi-crystallin by DTNB was caused by a modification of one cysteine residue per subunit. The resulting DTNB-modified enzyme was reactivated by dithiothreitol (DTT) or KCN. The inactivation was partially protected by NADPH, whereas 9,10-phenanthrenequinone (PQ) enhanced the modification of the enzyme. These results indicate that the SH- group being modified is located at/near the NADPH binding site which is not essential for catalysis. Incubation of the enzyme with DTT led to a substantial loss of the enzyme activity. However, DTT inhibition was prevented completely by preincubation of enzyme with PQ but not by NADPH indicating that an essential disulfide-bridge is present at the substrate binding site of xi-crystallin.

Inhibition kinetics of camel lens zeta-crystallin: multiple inhibition studies.

The inhibition of camel lens zeta-crystallin by nitrofurantoin (NF) was uncompetitive with respect to co-factor NADPH, (Ki = 90 microM) and competitive with respect to the substrate 9,10-phenanthrenequinone (PQ), (Ki = 50 microM). Inhibition at micromolar concentrations was also observed with dicoumarol, NADP+ and cibacron blue (CB). Theorell-Yonetani double-inhibition analysis showed that NF and dicoumarol were mutually exclusive inhibitors against PQ. However, analysis of NF and NADP+ by a double-inhibition plot showed that they simultaneously bind to the enzyme molecule. These studies demonstrate that NF and dicoumarol share the same site so that both molecules are prevented from binding at the same time, while NF and NADP+ can bind simultaneously to different sites on the enzyme. Although CB was noncompetitive with respect to PQ, double inhibition analysis showed that CB and dicoumarol or NF were mutually exclusive inhibitors against PQ, implying a distinct mode of inhibition for CB.

Age-dependent variations in the camel lens crystallins.

1. Water-soluble crystallins from cortex and nucleus of both young and adult camel lenses were fractionated by gel filtration into high molecular weight aggregate (HMW-aggregate), alpha-low, beta-high, beta-low, gamma-high and gamma-low protein fractions. 2. Crystallins were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Changes related to age and lens parts were compared. 3. The major changes in crystallin distribution included an increase in HMW-aggregate (alpha-high), beta-high, and gamma, and a decrease in alpha-low and beta-low, when comparing lenses in the direction of the nucleus and in the direction of increasing age. 4. Similar changes were detected comparing either cortex and nucleus of the same lens or whole lenses of different age.

Isolation of brain alpha-actinin. Its characterization and a comparison of its properties with those of muscle alpha-actinins.

A rapid purification procedure has been developed for the isolation of alpha-actinin from chicken brain. Brains were homogenized in cold water containing 0.5 mM phenylmethanesulfonyl fluoride (PMSF), the homogenate was centrifuged, and the alpha-actinin was extracted from the pelleted material in a low ionic strength buffer for 30 min at 22 degrees C. Purification of the protein to homogeneity on sodium dodecyl sulfate containing polyacrylamide gels required an ammonium sulfate precipitation step followed by chromatography on columns of DEAE-cellulose, hydroxylapatite, and Sepharose CL-6B. The alpha-actinins from chicken pectoral muscle (skeletal) and gizzard (smooth muscle) were purified in a similar fashion but without the DEAE-cellulose chromatography step. All three alpha-actinins have an identical Stokes radius of 7.1 nm determined by gel filtration chromatography. The individual proteins are homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but do not comigrate; however, all three alpha-actinins have identical retardation coefficients, obtained from electrophoretic mobilities at different acrylamide concentrations, which indicates that they all have similar subunit molecular weights (about 105 000). All three proteins behave similarly on isoelectric focusing gels (pI of native proteins congruent to 4.7-4.9) and have similar UV and CD spectroscopic properties. Significant differences exist both in their amino acid composition and in their peptide maps, obtained from limited proteolysis, which indicates that the proteins are all unique gene products.(ABSTRACT TRUNCATED AT 250 WORDS)