Enzyme complex structure and location of enzymes in the phthalate degradative pathway in Rhodococcus erythropolis

A. SUEMORI, K. NAKAJIMA, R. KURANE AND Y. NAKAMURA. 1996. Rhodococcus erythropolis strain S1 formed enzymes essential to the degradation of phthalate when grown in phthalate-minimal medium. The reaction responsible for the dihydroxylation of the phthalate-benzene ring was concluded to be catalysed by membrane-associated phthalate 3,4-dioxygenase (PO). Of the other enzymes involved, 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH) and 3,4-dihydroxyphthalate 2-decarboxylase (PC) appeared likely to be membrane-bound, while protocatechuate 3,4-dioxygenase appeared to be present in the cytoplasm. Based on the data, the membrane-bound PO and PH apparently form an enzyme complex, which is associated with the NADH-regenerating system.     

The transformation of phthalaldehydate by phthalate-grown Micrococcus strain 12B

Micrococcus strain 12B, grown with phthalate, transformed the phthalate analog, phthalaldehydate (2-formylbenzoate), to 3,4-dihydroxyphthalaldehydate which was isolated and identified as its lactol. An ¹⁸O₂ incorporation experiment indicated that a dioxygenase mechanism was involved. It is proposed by analogy, that phthalate is metabolized through cis-3,4-dihydro-3,4-dihydroxyphthalate and 3,4-dihydroxyphthalate by this bacterium.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.     

Biodegradation of Phthalate Isomers by Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4

Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.     

Utilization of phthalate esters by micrococci

Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source. They have been separated into four groups by their esterase content and nutritional characteristics. The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups. Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.     

A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms

We have developed a simple method for the detection of phthalate 4,5-dioxygenase and 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase activities in the initial step of phthalate degradation in bacteria. It was found that cells of a Pseudomonas putida strain adapted for phthalate could convert quinolinic acid to a hydroxylated product having λ_max at 315 nm. The occurrence of this compound was visualized by reaction with diazotized p-nitroaniline with which a red compound having λ_max at 512 nm was produced. In practice, if cells in colonies developed on an agar plate containing mineral salt medium supplemented with 0.4% of disodium phthalate and 0.1% of quinolinic acid are active with respect to the 4,5-dihydroxyphthalate pathway, then the colonies would be colored red immediately upon spraying with the diazotized p-nitroaniline reagent. The method was used to identify the phthalate degradative pathway for 27 phthalate-utilizing strains of the genera Pseudomonas (18 strains), Agrobacterium (3 strains), Alcaligenes (5 strains), and Micrococcus (1 strain). It was found that 24 of the 26 Gram-negative bacteria have the 4,5-dihydroxyphthalate pathway and that the remaining two strains of Pseudomonas sp. may metabolize via an unidentified pathway other than the dihydroxyphthalate pathways, and the Gram-positive strain of Micrococcus sp. metabolizes phthalate via the 3,4-dihydroxyphthalate pathway.     

Surface Glycolipids in Shoot-forming Culture of Nicotiana umbratica

Phthalate oxygenase was induced in Rhodococcus erythropolis S-1, a Gram-positive bacterium, when this bacterium was cultured in a medium containing phthalate as a sole carbon source. The enzyme was purified 118-fold with 4.7% activity yield. The purified enzyme appeared homogenous on native PAGE. This enzyme is a large protein (213 kDa), a tetramer of identical 56kDa monomers, and a flavoprotein containing FAD with NADH-dependent dioxygenase activity. The enzyme is specific for phthalate and other closely related aromatic compounds. Optimum pH and temperature were 6.5 and 40°C. The K_m for phthalate and NADH were 0.040 mM and 0.069 mM. The enzyme catalyzes dihydroxylation of phthalate to form 3,4-dihydro-3,4-dihydroxyphthalate with consumption of NADH and oxygen.     

Production of 4,5-dihydro-4,5-dihydroxyphthalate from phthalate by a mutant strain of Pseudomonas testosteroni M4-1

Summary Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate. Offprint requests to: T. Omori     

Subcellular Distribution of Basal and Cation-sensitive ATPases in Roots of Phaseolus vulgaris

A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg²⁺, Na⁺ and K⁺, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane.     

Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-octaprenyl-4-hydroxybenzoate carboxy-lyase

A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lyase was shown to require Mn²⁺, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.     

Isolation and characterization of marine bacteria capable of utilizing phthalate

Eleven phthalate-degrading bacterial strains were isolated from seawater collected off the coast of Japan. The isolates were found to be most closely related to the marine bacterial genera Alteromonas, Citreicella, Marinomonas, Marinovum, Pelagibaca, Rhodovulum, Sulfitobacter, Thalassobius, Thalassococcus, Thalassospira, and Tropicibacter. For the first time, members of these genera were shown to be capable of growth on phthalate. The plate assay for visual detection of phthalate dioxygenase activity and PCR detection of a possible gene encoding 4,5-dihydroxyphthalate decarboxylase indicated that phthalate is degraded via 4,5-dihydroxyphthalate to protocatechuate in all the isolates.     

Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.     

Body Plasma Membrane Vesicles from Paramecium Contain a Vanadate-Sensitive Ca--ATPase

Paramecia are an excellent model system for studying the mechanisms involved in sensory transductions and intracellular Ca²⁺ regulation. These cells have two functionally distinct plasma membrane domains, body and cilia. The body plasma membrane is responsible for transduction of sensory stimuli into receptor potentials and the ciliary membrane is required for Ca²⁺ action potentials. Although ciliary membrane vesicles (cmv) have been purified and well characterized, body plasma membranes have not. We have generated body plasma membrane vesicles (bmv) by homogenization of deciliated cells and purified them from the microsome fraction by a two-phase aqueous polymer separation. The major criteria for purity of the bmv fraction are: (i) It is enriched 15-fold for a known plasma membrane marker (immobilization antigen) while the marker activities for other membranes were all decreased. The protein banding pattern of bmv is generally similar to cmv on SDS-PAGE. (ii) It contains a vanadate-sensitive Ca²⁺-ATPase activity that has been suggested to be a plasma membrane Ca²⁺ pump. The specific activity of this bmv Ca²⁺-ATPase is increased 4-fold over that of the homogenate. (iii) The phospholipid, fatty acid, and sterol composition of the bmv fraction are indicative of plasma membranes because they are qualitatively similar to cmv. The bmv also contains a membrane-bound NADPH-dependent cytochrome c reductase activity, suggesting that it may play a role in body plasma membrane function. This purified bmv preparation is useful for studying the role of the body plasma membrane in Ca²⁺ regulation, sensory transduction, protein and lipid trafficking, and plasma membrane fusion events.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

Energy-Dependent Ca2+ Efflux from the Cells of Nostoc calcicola Bréb: Role of Modifying Factors

Energy-dependent Ca²⁺ efflux and its regulation from the diazotrophic cyanobacterium Nostoc calcicola Bréb has been investigated. Like Ca²⁺ uptake, Ca²⁺ efflux pattern also reflected a rapid phase for the first 10 min followed by a slower one lasting up to 1 h with a total of 80 nmol Ca²⁺ mg⁻¹ protein (31% of the Ca²⁺ concentration taken in by such cells at 1 h). Ca²⁺ efflux kinetics remained hyperbolic with a K _m of 1.9 mM and V_max 5.5 nmol mg⁻¹ protein min⁻¹. Ca²⁺ efflux to a major extent depended on photosynthetic energy generation as the cells facing dark incubation and addition of 3-(3,4-dichlorophenyl)-1-dimethyl urea (DCMU) to light-grown cells showed significant reduction in Ca²⁺ extrusion. The strong inhibition in Ca²⁺ efflux by addition of metabolic inhibitors like carbonyl cyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP) and N,N,-dicyclohexylcarbo-diimide (DCCD) suggested the vital role of membrane potential and ATP hydrolysis in driving this process. Verapamil (Ca²⁺ antagonist) had insignificant effect on Ca²⁺ efflux, whereas the addition of Calmodulin antagonists like trifluoroperazine, W-7 and compound 48/80 resulted in the enhancement in Ca²⁺ efflux over control sets, thus suggesting that this increase may be owing to the additional extrusion of intracellular free calcium that was unable to bind with calmodulin in the presence of these antagonists. Received: 30 April 1999 / Accepted: 22 May 1999     

A comparative study of 5’nucleotidase and alkaline phosphatase in human placenta during development

Activities and a few properties of alkaline phosphatase and 5’-nucleotidase were compared in the developing human placenta. Both the enzymes were mostly membrane-bound and displayed similar developmental patterns with the highest activities at 24/26 weeks of the placenta. L-Phenylalanine, L-tryptophan and L-leucine were inhibitors of alkaline phosphatase, whereas they had no effect on the 5’-nucleotidase. Alkaline phosphatase from a late stage of gestation appeared to be almost heat-stable. An appreciable part of 5’-nucleotidase was also resistant to heat inactivation and this fraction varied with gestational age of the tissue. For both the enzymes, V_max changed without alteringK _m values with periods of gestation. Ca²⁺, Mg²⁺ and Mn²⁺ ions stimulated the alkaline phosphatase activity and Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ were inhibitory. 5’-Nucleotidase was not activated by any of these cations. EDTA and Concanavalin A inhibited both the enzymes, although the extent of inhibition was different and also varied with gestation.     

Purification and characterization of phenoloxidase from the hemolymph of healthy and diseased Antheraea assamensis Helfer (Lepidoptera: Saturniidae): Effects of certain biological components and chemical agents on enzyme activity

In the current study, a dimeric phenoloxidase (PO) from the hemolymph of healthy and diseased (pebrine infected) larvae of Antheraea assamensis Helfer was extracted and purified. The protein was subjected to purification using Sephacryl S‐100 and CM Sepharose chromatography. The enzyme comprised of two subunits of ~76.8 and 76 kDa that showed PO activity in 6 mM l ‐3,4‐dihydroxyphenylalanine (L ‐DOPA) and 8 mM catechol but not in hydroquinone. Optimum temperature for PO activity was 30°C in l ‐DOPA and 37°C in catechol. Optimum pH ranged from 6.8 to 7.0 in L ‐DOPA and 7.0–7.2 in catechol. Specific activity of the purified PO from healthy larvae was 53.9 µM/min per mg of protein per ml in L ‐DOPA and 50.77 µM/min per mg of protein per ml in catechol. Specific activity of PO from diseased larvae was 30.0 µM/min per mg of protein per ml in L ‐DOPA and 28.55 µM/min per mg of protein per ml in catechol. Purification fold was 3.27–4.21 for healthy and 2.38–2.56 for diseased fractions. The enzyme showed the Michaelis constant (K_m) of 2.46–2.85 mM for healthy and diseased fractions in L ‐DOPA. In catechol K_m of 9.23–17.71 mM was observed. Peptidoglycan was the best activator of purified PO from both healthy and diseased fractions. Interactions between controls and activators appeared statistically significant (F = 767.5; df = 3; P < 0.0001). Na⁺, K⁺, and Cu²⁺ increased, whereas Ca²⁺, Zn²⁺, Mg²⁺, and Co²⁺ decreased PO activity. The overall interactions appeared highly significant (F = 217.0; df = 27; P < 0.0001). Kojic acid, dithiothreitol, thiourea, phenylthiourea, carbendazim, N‐bromosuccinimide, N,N,N′,N′‐tetraacetic acid, and diethyldithiocarbamate inhibited PO activity.