Evaluation of Natural Additives to Enhance the Persistence of Spodoptera littoralis (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (SpliMNPV) Under Field Conditions in Saudi Arabia.

Nucleopolyhedrovirus is an effective biocontrol agent but for its biggest disadvantage of short persistence under sunlight conditions. In this study, 10 plant extracts were evaluated as ultraviolet (UV) protectants to improve the persistence of Spodoptera littoralis multiple-embedded nucleopolyhedrovirus (SpliMNPV) against cotton leafworm (Spodoptera littoralis Boisduval). In the primary lab screening test, 5 out of 10 additives (cloves, henna, green tea, pomegranate, and grape extracts) presented a high rate of virus protection with original activity remaining (OAR) percentage of 100%, 97%, 91%, 90.6%, and 77%, respectively, when used at a concentration of 1% and exposed to UVB for a period of 1 h. A secondary screening was then performed with these best five extracts at a concentration of 0.5% and for an exposure timing of 5 h to UVB. Among these, clove and henna that showed highest protection with OAR values of 96.6% and 76.5%, respectively, were selected for the field trials. When applied on cabbage in the field during sunny summer conditions, clove and henna extracts enhanced the persistence of SpliMNPV by twofold. These findings are encouraging to be applied in the field studies.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

BACKGROUND: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. RESULTS: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. CONCLUSIONS: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Combined effect of nucleopolyhedrovirus and Microplitis pallidipes for the control of the beet armyworm, Spodoptera exigua.

BACKGROUND: Nucleopolyhedrovirus (SeNPV) and Microplitis pallidipes are important biological control agents of Spodoptera exigua populations. The interactions between these agents and their combined effect on pest control were investigated in the laboratory and in commercial greenhouses. RESULTS: Microplitis pallidipes searched for and deposited eggs in more healthy larvae than virus-infected larvae 3 days after viral infection. Each female parasitoid that developed in a virus-infected host oviposited in a virus-infected host, or emerged from a cocoon carrying virus transmitted to 4.0, 7.6 or 2.4 healthy larvae respectively. Each female parasitoid exposed to a mixture of virus and 10% honey water solution transmitted the virus to 2.2 healthy larvae. In an experiment with cabbage growing in commercial greenhouses, the pest population reduction was greater by M. pallidipes carrying SeNPV (82.3-89.7% reduction) than by parasitoids without virus (59.5-62.4% reduction). CONCLUSIONS: Control of S. exigua was greater with M. pallidipes plus SeNPV than with M. pallidipes alone. Microplitis pallidipes preferred healthy hosts to infected hosts. Parasitoids were able to complete their development in virus-infected hosts before the hosts died from the virus infection. The parasitoid ovipositors contaminated with the virus could carry and transmit SeNPV.

Tea, coffee, and cocoa as ultraviolet radiation protectants for the beet armyworm nucleopolyhedrovirus.

The addition of 1% (wt:vol) aqueous extracts of cocoa (Theobroma cacao L.) (Malvales: Malvaceae), coffee (Coffea arabica L.) (Gentianales: Rubiaceae), and green and black tea (Camellia sinensis L.) (Ericales: Theaceae) provided excellent UV radiation protection for the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), nucleopolyhedrovirus under laboratory conditions. Aqueous extracts of coffee, green tea, and black tea at 0.5% provided 85-100% UV protection, whereas cocoa provided 50% UV protection. Epigallocatechin gallate (EGCG), a component of green tea, and caffeine, a component of tea and coffee, also were tested as UV protectants. Both compounds were ineffective when tested alone. When EGCG and caffeine were combined, UV protection increased in a synergistic manner, but <35% of the original virus activity was maintained. This study demonstrated that coffee was comparable to green tea and black tea as a UV protectant. Further studies should be conducted to optimize their use in biopesticide formulations.

Display of a maize cDNA library on baculovirus infected insect cells.

BACKGROUND: Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. RESULTS: We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. CONCLUSION: The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

[Complex formation between recombinant protein VPg of potato virus Y and heterologous eukaryotic translation initiation factors eIF4E].

Genomes of some positive-strand RNA viruses do not contain cap-structure, but instead their 5'-end is covalently linked to a viral protein called VPg. Complex formation between VPg and cellular translation initiation factors (eIFs) has been extensively studied in the context of the model of this complex involvement in virus mRNA translation initiation and cellular protein translation shut down in infected cells. The potato virus (PVY) VPg was expressed in bacterial and baculovirus systems in order to investigate its binding capacity to wheat eIF4E and its isoform. Both purified recombinant eIF4E and eIF(iso)4E were identified in vitro as binding partners of the purified recombinant VPg by using affinity chromatography, as well in vivo by coexpressing of recombinant VPg and eIFs in insect cells with following complex purification using affinity chromatography. Besides it was shown that PVY VPg also formed a complex with endogenous insect eIF4E in vivo. PVY VPg interaction with eIF4E of wheat (non permissive plant for PVY), and also with so evolutionary distant partner as insect eIF4E suggests the conservation of general structural features of eIF4E implicated in the formation of the complex with VPg.

Biochemical characterization of Sf9 Sp-family-like protein factors reveals interesting features.

We earlier documented the involvement of novel Sp-family-like protein factors in transcription from the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin (polh) gene promoter [Ramachandran et al. (2001) J. Biol. Chem. 276: 23440-23449]. These zinc-dependent Sp-like factors bind to two putative Sp-factor-binding motifs, present within the AcSp sequence upstream of the polh promoter, with very high affinity (K(d) = 2.1 x 10(-12) M). Like other polh-promoter-associated host transcription factors, these Sp-like factors display tolerance to high ion concentrations up to even 3 M NaCl. An electrophoretic mobility shift assay demonstrated a probable cross-talk between the Spodoptera frugiperda (Sf9) Sp-family-like proteins and the TFIID complex. In complementary experiments, specific replacements of the Sp-factor-binding motifs with TATA-like elements resulted in expression of a luciferase reporter gene to almost the same level as that obtained with a wild-type native construct. Our results point to the possibility of the involvement of TFIID and Sf9 Sp protein interaction in transcription from the baculovirus polyhedrin promoter.

Determination and characterization of bovine interleukin-17 cDNA.

Interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated memory T cells, and it appears to play an upstream role in T cell-triggered inflammation by stimulating stromal cells to secrete other cytokines. We hypothesize that IL-17 plays a role in the recruitment of neutrophils in the bovine mammary gland during infection or immune-mediated inflammation. The rapid amplification of cDNA ends (RACE) method was used to obtain a cDNA of bovine IL-17 (BoIL-17) containing a 462-bp open reading frame (ORF) encoding a protein of 153 amino acids (aa) with a molecular mass of 17.2 kDa, a 23-residue NH(2)-terminal signal peptide, a single potential N-linked glycosylation site, and 6 cysteine residues. BoIL-17 protein shared 73.5% identity with the human protein and 67% with the mouse and rat proteins. Sf9 insect cells were transfected with BoIL-17 cDNA, and supernatant was tested for biologic activity on a primary culture of bovine mammary epithelial cells (MECs). mRNA synthesis of IL-6, IL-8, and growth-related oncogene alpha (Groalpha) was induced, suggesting a functional role for IL-17 in mammary immunity.

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions.

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.

St. John's wort extracts and some of their constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1.

Commercially available St. John's wort (Hypericum perforatum) preparations and some of their main constituents (hypericin, pseudohypericin, hyperforin, rutin, and quercetin) were examined for their potential to inhibit carcinogen activation by human cytochrome P450 1A1 (CYP1A1). We used a reconstituted system consisting of purified human CYP1A1, purified human NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine as lipid component. St. John's wort extracts potently inhibited CYP1A1-catalyzed (+/-)-trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (7,8-diol-B[a]P) epoxidation, the terminal reaction leading to the ultimate carcinogenic product (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2). All constituents, except rutin, were shown to possess strong inhibitory potencies toward diolepoxide 2 formation from 7,8-diol-B[a]P, with IC(50) values of 0.5 microM (hypericin), 1.2 microM (hyperforin), 1.5 microM (quercetin), and 8 microM (pseudohypericin), respectively. Preincubation experiments revealed that their action was not mechanism based. Inhibition kinetics studies showed the anthrodianthrone compound hypericin to be a noncompetitive inhibitor, with a K(i) value of 0.6 microM, and the phloroglucinol hyperforin to be a competitive inhibitor, with a K(i) value of 1.1 microM. When the effects on NADPH-P450 reductase activity were investigated, all constituents of St. John's wort studied turned out to be rather ineffective inhibitors; quercetin was the only exception, with an IC(50) value of approximately 20 microM. These in vitro data indicate that St. John's wort extracts and some of their constituents potently inhibit the major human procarcinogen-activating enzyme CYP1A1.

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system.

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.

Sensitivity of Invertebrate iridescent virus 6 to organic solvents, detergents, enzymes and temperature treatment.

The sensitivity of Invertebrate iridescent virus 6 (IIV-6) to a selection of organic solvents, detergents, enzymes and heat treatment was assayed in Spodoptera frugiperda (Sf9) cells and by injection of inoculum into larvae of Galleria mellonella. In several cases, the degree of sensitivity of the virus depended on the method of assay; cell culture assays indicated greater losses of activity than insect bioassay. IIV-6 was sensitive to chloroform but sensitivity to ether was only detected by cell culture assay. Sensitivity (defined as a reduction of at least 1 log activity) was detected following treatment by 1 and 0.1% SDS, 1% Triton-X100, 70% ethanol, 70% methanol, 1% sodium deoxycholate, pH 11.1 and 3.0. No sensitivity was detected to 1% Tween 80, 1 M MgCl2, 100 mM EDTA, lipase, phospholipase A2, proteinase K, or trypsin at the concentrations tested. Viral activity was reduced by approximately 4 logs following heating to 70 degrees C for 60 min or 80 degrees C for 30 min. The above observations highlight the need for studies on the role of the virus lipid component in the process of particle entry into cells, and may explain why vertebrate and invertebrate iridoviruses have been reported to differ in their sensitivity to organic solvents and enzymes.

SULT1A1 catalyzes 2-methoxyestradiol sulfonation in MCF-7 breast cancer cells.

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species.

Eukaryotic initiation factor eIF5A is essential for cell viability and contains a characteristic post-translational modification of a specific lysine residue into a hypusine. cDNAs with similarity to eIF5A sequences were derived from Spodoptera exigua and S. frugiperda cDNA libraries. The deduced amino acid sequences are identical for both species and predict a protein with a molecular mass of 17.5 kDa. The Drosophila melanogaster eIF5A cDNA sequence was retrieved from the Drosophila EST Project. The predicted protein is 80% similar to Spodoptera eIF5A. A single eIF5A gene copy is present in the S. frugiperda genome, which is transcribed into four different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decline of all four transcripts already at 12 h after infection. In contrast, the eIF5A protein was fairly stable up to 48 h post infection.

Differential requirements of two insect cell lines for growth in serum-free medium.

The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.

Multiplication of Spodoptera littoralis granulosis virus in a cell line established from Phthorimaea operculella.

A complete replication of the Spodoptera littoralis granulosis virus (SpliGV) was obtained, in vitro by both virus infection and DNA transfection in the ORS-Pop-95 (Pop-95) cell line established from embryonic cells of the potato tuber moth, Phthorimaea operculella. SpliGV multiplied significantly during several passages in Pop-95 cells at 19 degrees C. When the cells were infected and kept at 19 degrees C for the first 4 hrs and then at 27 degrees C for the rest of the experiment (20 days), the viral multiplication proceeded at the same rate. Comparison of SpliGV progenies, multiplied either in vivo or in vitro, using electron microscopy and restriction profile analysis, showed their identity.

Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding.

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine into L-citrulline and nitric oxide. Recently we have developed a method for expression of recombinant rat brain NOS in baculovirus-infected Sf9 cells and purification of the enzymically active enzyme [Harteneck, Klatt, Schmidt and Mayer (1994) Biochem J. 304, 683-686]. To study how biosynthetic manipulation of the NOS cofactors haem, FAD/FMN, and tetrahydrobiopterin (H4biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 microg/ml), riboflavin (0.1.mM), and the inhibitor of H4biopterin biosynthesis 2,4-diamino-6-hydroxypyrimidine (10 mM). In the absence of haemin, NOS was expressed to a very high level but remained predominantly insoluble. Purification of the soluble fraction of the expressed protein showed that it had poor activity (0.35 micromol of citrulline x mg(-1) x min(-1)) and was haem-deficient (0.37 equiv. per monomer). Supplementing the culture medium with haemin resulted in pronounced solubilization of the expressed enzyme, which had a specific activity of approximately 1 micromol of citrulline x mg(-1) x min(-1) and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H(4) biopterin endogenously present in the different NOS preparations positively correlated with the amount of enzyme-bound haem (y = 0.066+0.430x; r = 0.998). Radioligand binding experiments demonstrated that haem-deficient enzyme preparations containing 30-40% of the holoenzyme bound only approximately 40% of H4biopterin as compared with haem-saturated controls. These results suggest that the prosthetic haem group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin.

Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides.

A full-length cDNA for a novel isoform of the human receptor tyrosine phosphatase gamma gene (PTPRG) was overexpressed in Sf9 insect cells, and the gene product, PTP gamma, was purified and characterized. The protein was expressed as a M(r) approximately 185,000 protein accompanied by a M(r) approximately 120,000 putative cleavage product on SDS-PAGE analysis. The protein undergoes N-linked glycosylation and constitutive phosphorylation of serine residues. When assayed for tyrosine-specific phosphatase activity, PTP gamma dephosphorylated myelin basic protein at a pH optimum of 7.5 and a Km of 12.6 microM; reduced carboxyamidomethylated and maleylated lysozyme (RCM-lysozyme) at a pH optimum of 6.0 and a Km of 12 microM; and p-nitrophenylphosphate with a pH optimum of 5.5 and a Km of 3.5 mM. Phosphatase activity was inhibited by ZnCl2 and sodium orthovanadate; Mg2+, Mn2+, and Ca2+ ions were ineffective. The partially purified form of the enzyme was allosterically activated by triphosphorylated nucleosides, with a preference for purines. This activation was prevented by Mg2+ addition and did not occur when a purified form of the enzyme was utilized, suggesting that its activation depends on specific activating factors or conformational constraints. Interestingly, PTP gamma protein was specifically bound by an ATP-agarose matrix through its intracellular domain, suggesting a link between binding of nucleotides and activation of the phosphatase.

Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase.

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5' and 3' ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3'-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.