Beta-naphthyl Esters Research Articles

Esterase-3 polymorphism in the sugarcane borer Diatraea saccharalis (Lepidoptera, Pyralidae)

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations.

Differential esterase expression in developmental mutants of Aspergillus nidulans.

Esterase isozymes were used to detect substrate-preference polymorphism in five strains of Aspergillus nidulans, and to show differential gene expression in developmental mutants in response to 5-azacytidine treatment. The medusa mutants B116, SM23, and M25 were selected in the presence of 5-azacytidine (5AC); also the G839 bristle mutant obtained in the absence of 5AC as well as the UT196 master strain and the normal segregant SM24 were used for the esterase studies. The esterase isozyme patterns of the A. nidulans strains observed with 4-methylumbelliferyl esters and alpha- and beta-naphthyl esters indicated a total of 18 isoesterases. Substrate preference for either 4-methylumbelliferyl esters and alpha- or beta-naphthyl esters was observed. Similarity between the different A. nidulans genotypes was 84.4-100%. The genomic similarity of the B116, SM23, and M25 mutant strains (100%) supports previous observations that specific DNA sequences might be targets for 5AC action in this filamentous fungus, and the differential expression of the Est-4 isozyme in the medusa developmental mutant and the Est-2 isozyme specifically detected in the bristle mutant G839 seems to indicate esterase isozymes as possible markers of biochemical differences among different developmental mutants of A. nidulans.

Mutations affecting a regulated, membrane-associated esterase in Salmonella typhimurium LT2.

Mutations at the apeA locus in Salmonella typhimurium lead to loss of a soluble enzyme ("protease I") that hydrolyzes the chromogenic endoprotease substrate N-acetyl phenylalanine beta-naphthyl ester. We have isolated pseudorevertants of S. typhimurium apeA mutations that have regained the ability to hydrolyze this compound. These pseudorevertants contain mutations (apeR) that lead to overproduction of a membrane-bound esterase different from protease I. The apeR locus is phage P1 cotransducible with ilvC (83 map units) and is unlinked to apeA. Mutations at still another locus, apeE, lead to loss of the membrane-associated esterase. The apeE locus is P1 cotransducible with purE (12 map units). In an apeE-lacZ operon fusion strain, an apeR mutation increases the level of beta-galactosidase approximately 60-fold. We propose that apeR encodes a repressor of apeE. The evidence available suggests that the ApeE protein is not a protease.

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

Esterase 6 from Drosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directed in vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for beta-naphthyl esters and p-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb's free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants

Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester. The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined. The proteinase was purified to homogeneity from the soluble fraction of an overproducing strain possessing the cloned gene. N-terminal amino acid sequencing of the purified protein revealed that the cloned gene is the structural gene for the protein, with the protein being synthesized in precursor form with a signal peptide. On the basis of its molecular mass (20 kDa), periplasmic localization, and substrate specificity, we conclude this protein to be protease I. By using the gene cloned on a plasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene (Kmr) on the chromosome. The Kmr gene was mapped at 11.8 min, the gene order being dnaZ-adk-ush-Kmr-purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonella typhimurium. Therefore, the gene was named apeA. Deletion of the apeA gene, either with or without deletion of other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the various media tested.

Substrate specificity of purified rabbit liver esterase ES-1.

1. Substrate hydrolysis by two purified rabbit liver esterase-1 allozymes (ES-1A and ES-1B) was compared under conditions differing in substrate, pH and temperature. ES-1A and ES-1B activities had a similar pH and temperature dependency and similar thermal stability profile. 2. There were marked differences in specific activity of ES-1A and ES-1B. ES-1A hydrolysed procaine more rapidly than ES-1B, but was less active towards aspirin. The acetate and propionate esters of p-nitrophenyl were hydrolysed slower by ES-1A than by ES-1B. 3. The effect of substrate concentration on ES-1A activity did not comply with the Michaelis-Menten kinetics, which may be due to so-called substrate activation. 4. At identical substrate concentration, pH and temperature, selected artificial esters were better substrates for ES-1A than selected physiological substrates. Beta-Naphthyloctanoate was found to be a suitable substrate for ES-1A. 1,3-Dioctanoylglycerol was hydrolysed at a rate of only 2% of that of beta-naphthyloctanoate. 5. With methyl, p-nitrophenyl, beta-naphthyl and 4-methylumbelliferyl esters as substrates, ES-1A activity is influenced by length and structure of the acyl moiety. Likewise, ES-1A activity is influenced by the nature of the alkyl moiety of acetate esters. With acetate and methyl esters, branched chains when compared with unbranched chains reduced the esterase activity of ES-1A. Elongation of the acyl moiety up to four or five C atoms gradually raised the velocity of methyl, p-nitrophenyl, and 4-methylumbelliferyl ester hydrolysis by ES-1A. A similar pattern was found for the length of the alkyl moiety of acetate esters. 6. The high degree of similarity between the observed substrate specificity of rabbit ES-1A and that reported earlier for rat ES-10, suggests that these two esterases have a common evolutionary origin.

Hydrolyses of α-naphthyl acetate, β-naphthyl acetate, and acetyl-DL-phenylalanine β-naphthyl ester

Using simultaneous coupling azo dye techniques kidney enzymes active against alpha-naphthyl acetate, beta-naphthyl acetate, and acetyl-DL-phenylalanine beta-naphthyl ester are characterized. The enzymes show identical distribution in the section. The banding patterns in zymograms are the same after incubation with the different substrates. The enzymes might, however, be separated by difference in pH optimum, initial velocity and sensitivity to inhibitors and activators.

Identification and localization of two membrane-bound esterases from Escherichia coli.

Hydrolytic activities of isolated membrane fractions of Escherichia coli against chromogenic substrates, p-nitrophenyl ester and beta-naphthyl ester derivatives of N-substituted amino acids, were investigated by spectrophotometric and electrophoretic methods. Although detergents were absolutely necessary for the solubilization of enzymes, the amount of solubilized activities was increased by adding salt, such as NaCl or KCl. Two esterases were identified and separated by PAGE and by chromatography of the solubilized proteins in the presence of detergent. One hydrolyzed the alanine derivatives preferentially, whereas the other was mainly active on phenylalanine derivatives. Only the first was inactivated by diisopropyl fluorophosphate, a serine hydrolase inhibitor. Whereas the chymotrypsin-like enzyme was equally distributed between the inner and the outer membrane, the alanine activity was only detected in the inner membrane. They were both resistant to extraction with high salt concentrations, indicating their integral association with membranes. A study of the accessibility of these enzymes to their substrate in membrane vesicles with known polarity suggests that both alanine and phenylalanine activities are localized near the external surface of the cytoplasmic (inner) membrane. However, the phenylalanine activity (chymotrypsin-like enzyme) appears to be deeply buried inside the outer membrane. Because of its insensitivity to diisopropyl fluorophosphate, this last esterase seems to be distinct from the previously isolated periplasmic endopeptidase, protease I, which is also a chymotrypsin-like enzyme.

Salmonella typhimurium mutants lacking protease II

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.

Acylaminoacid esterase mutants of Salmonella typhimurium

Salmonella typhimurium contains three electrophoretically separable enzyme activities that hydrolyze N-acetyl phenylalanine beta-naphthyl ester (NAPNE). One of these enzymes is an endoprotease, protease I. Mutations at a locus apeA near purE lead to loss of this enzyme. We have found that N-acetyl leucine alpha-naphthyl ester (NALNE) is not hydrolyzed by protease I but is a good substrate for the other two activities. Using NALNE as a chromogenic substrate to screen colonies growing on agar, we have isolated mutants (apeB) that simultaneously lose both of the two other esterase activities. The chromosomal positions of apeB and nearby markers in the proC-purE region have been determined using both phage P1 and phage P22 mediated transduction. The observed order is proC thiC apeB apt apeA purE. Strains lacking all three activities (apeA apeB double mutants) have been constructed and have growth rates similar to wild-type strains.

Relationships between electrophoretic patterns of esterases from Salmonella.

Esterases of 85strains of the four biochemically-defined subgenera of Salmonella, when analysed by the acrylamide-agarose zymogram technique using several synthetic substrates, gave four principal bands (E1, E2, E3, E4) and two minor ones. The E1 esterase band hydrolysed alphs-naphthyl acetate, whereas the E2 band hydrolysed beta-naphthyl acetate. These bands were resistant to di-isofluoropropyl phosphate (DFP) and their electrophoretic distribution among the strains occurred within a relatively small MF range, MF being the distance moved by the esterase band as a percentage of the distance moved by the dye front. The E3 band hydrolysed alpha-naphthyl acetate and alpha-naphthyl napbuyyrate and, to a lesser degree, beta-naphthyl esters, whereas the E4 band hydrolysed alpha-naphthyl acetate. These bands were sensitive to DFP and their electrophoretic distribution among the strains occurred in a wide MF range. ALL Salmonella strains were closely related in terms of their esterase profiles. However, the divergences in electrophoretic distribution of bands E3 and E4 were sufficient to recognize the subgenera of most of the Salmonella strains analysed.

Mutants of Salmonella typhimurium deficient in an endoprotease.

Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.

Esterases of the polymorphonuclear leukocyte capable of hydrolyzing acetyl DL-phenyl-alanine beta-naphthyl ester. Relationship to the activatable esterase of chemotaxis.

Previous published work has led to the hypothesis that the activatable esterase of chemotaxis is a serine esterase of the rabbit polymorphonuclear leukocyte existing in an inert, phosphonate insusceptible form, which after activation is capable of hydrolyzing aromatic amino acid esters and being inhibited by phosphonates. In the present study, directed to the testing of this hypothesis, we have shown that rabbit peritoneal polymorphonuclear leukocytes contain three esterases capable of hydrolyzing the aromatic amino acid ester, acetyl DL-phenylalanine beta-naphthyl ester. Two of these esterases, esterase 1 and esterase 2, are inhibited by various p-nitrophenyl ethyl phosphonate esters. The inhibition of each esterase is irreversible and progressive with time. When the logarithm of the esterase activity remaining after cell and inhibitor have been in contact for a constant time is plotted against the concentration of inhibitor, a straight line results. These results support the conclusion that both esterases are serine esterases. The third esterase, esterase 3, differs from the other two by its inability to be inactivated by any of the phosphonates no matter how high the concentration of phosphonate or prolonged the period of incubation of cell with phosphonate. The activity of esterase 1 is at least 10,000 times more easily inhibited by phosphonates than is that of esterase 2; incubating rabbit polymorphonuclear leukocytes for 15 min at 27 degrees C with 10(-9)-10(-8)M concentrations of various phosphonates inactivates esterase 1, but it required 10(-6)-10(-4)M concentrations of the same phosphonates to inhibit esterase 2. The inhibition profiles of esterase 1 are distinctly different from those of esterase 2 when the two esterases are tested with the phenylalkylphosphonates, chloroalkylphosphonates, and alkylphosphonates. The inhibition profile of esterase 1 is essentially the same as that of the activatable esterase of chemotaxis obtained previously when the same three homologous series of phosphonates were tested for their ability to protect against deactivation by the chemotactic factor or give chemotactic-dependent inhibition. It is tentatively concluded that esterase 1 of the rabbit peritoneal neutrophil is the activated form of the activatable esterase of chemotaxis.