Hormonal control of tick development and reproduction.

Ecdysteroids (moulting hormones), juvenoids and neuropeptides in ticks are reviewed but, by far, the emphasis is on the former since this class of hormones has been the subject of most investigations. In immature stages of ticks, ecdysteroids have been shown to regulate moulting and to terminate larval diapause. Although there is a paucity of information on the molecular action of ecdysteroids in ticks, their action appears to be via a heterodimeric ecdysone/ultraspiracle receptor, as in insects. The role of ecdysteroids in sperm maturation in adult males is considered. In females, ecdysteroids function in the regulation of salivary glands, of production of sex pheromones and of oogenesis and oviposition. There is evidence for ecdysteroid production in the integument and pathways of hormone inactivation are similar to those in insects. Ecdysteroids also function in embryogenesis. Although evidence for the occurrence and functioning of juvenile hormones in ticks has been contradictory, in recent thorough work it has not been possible to detect known juvenile hormones in ticks, nor to demonstrate effects of extracts on insects. Factors (neuropeptides) from the synganglion affect physiological processes and limited immunocytochemical studies are reviewed. Sigificantly, a G-protein-coupled receptor has been cloned, expressed, and specifically responds to myokinins.

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus.

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Ecdysteroids and bufadienolides from Helleborus torquatus (Ranunculaceae).

Three bufadienolides, hellebortin A (5-[beta-D-glucopyranosyloxy]-10,14,16-trihydroxy-19-nor-[5beta,10beta,14beta,16beta]-bufa-3,20,22-trienolide [1]), hellebortin B (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-14-hydroxy-19-oxo-bufa-20,22-dienolide [2]) and hellebortin C (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-10,14-dihydroxy-19-nor-bufa-20,22-dienolide [3]), together with 20-hydroxyecdysone 3-O-beta-D-glucoside (4) and 20-hydroxyecdysone (5) have been isolated by bioassay- and RIA-directed HPLC analyses of a methanol extract of the seeds of Helleborus torquatus. The structure and relative stereochemistry of the novel bufadienolide hellebortin A (1) and the structures of hellebortin B (2) and hellebortin C (3) were determined unambiguously by comprehensive analyses of their 1D and 2D NMR data. These five compounds are isolated from Hellborus torquatus for the first time. The biological activities of compound 1, 4 and 5 as ecdysteroid agonists and antagonists have been assessed.

Regulation of ecdysteroid signaling: cloning and characterization of ecdysone oxidase: a novel steroid oxidase from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3alpha-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P)(+)-linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

Limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[1-->3]-beta-D-xylopyranoside), a phytoecdysteroid from seeds of Limnanthes alba (limnanthaceae).

A new ecdysteroid glycoside, limnantheoside C (20-hydroxyecdysone 3-O-beta-D-glucopyranosyl-[-->3]-beta-D-xylopyranoside [1]), together with limnantheoside A (20-hydroxyecdysone 3-O-beta-D-xylopyranoside [2]) and 20-hydroxyecdysone (3) have been isolated by bioassay/RIA-directed HPLC analyses of a methanol extract of the seedmeal of Limnanthes alba Hartw. ex Benth. The structure of the novel ecdysteroid glycoside (1) was determined unambiguously by UV, LSIMS and a combination of 1D- and 2D-NMR experiments. These three compounds are isolated from Limnanthes alba for the first time.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone.

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.

Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3 alpha-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3 alpha-reductase to 3-epiecdysone. The 3DE 3 alpha-reductase has been purified and subjected to limited amino acid sequencing. It occurs as two distinct forms, including a probable trimer of subunit molecular mass of approx. 26 kDa. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.2 kb) encoding the 26 kDa protein. Northern blotting showed that the mRNA transcript was expressed in Malpighian tubules during the early stage of the last larval instar. Conceptual translation of the 3DE 3 alpha-reductase cDNA and database searching revealed that the enzyme belongs to the short-chain dehydrogenases/reductases superfamily. Furthermore, the enzyme is a novel eukaryotic 3-dehydrosteroid 3 alpha-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3 alpha-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3 alpha-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3 alpha-reductase and a previously reported 3DE 3 beta-reductase, which acts on the same substrate and belongs to the AKR family.

Characterization of ecdysteroid 26-hydroxylase: an enzyme involved in molting hormone inactivation.

Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus.

Methyl farnesoate, the crustacean juvenoid, is synthesized and secreted from the mandibular organs of crustaceans under the negative control of the sinus gland-derived mandibular organ-inhibiting hormone (MO-IH). Previously we isolated and sequenced two isoforms, MO-IH-1 and MO-IH-2, differing by just one amino acid, from sinus glands of the edible crab, Cancer pagurus. We now report the isolation of cDNAs encoding MO-IH-1 and MO-IH-2 by a combination of reverse-transcriptase-mediated PCR in conjunction with 5' and 3' rapid amplification of cDNA ends ('RACE'). Full-length clones of MO-IH-1 and MO-IH-2 encoded a 34-residue putative signal peptide and the mature 78-residue MO-IH sequences. Northern blot analysis of various tissues showed that MO-IH expression is confined to the X-organ (a cluster of perikarya within the eye). Southern blot analysis indicated that there are approx. 10 copies of the gene for MO-IH in C. pagurus. Additional Southern blotting experiments detected MO-IH-hybridizing bands in another Cancer species, C. antennarius. In support of this, an HPLC-radioimmunoassay analysis of sinus gland extracts of C. antennarius and C. magister also revealed MO-IH-like immunoreactivity.

Involvement of adenosine cyclic-3',5'-monophosphate in the signal transduction pathway of mandibular organ-inhibiting hormone of the edible crab, Cancer pagurus.

The juvenoid, methyl farnesoate (MF), is synthesized in the mandibular organs (MOs) of crustaceans, under the control of mandibular organ-inhibiting hormone (MO-IH). Using an in vitro assay to measure synthesis of MF by MOs, the effect of a variety of agents that affect signal transduction pathways was investigated. Of the compounds tested, only agents which affect cAMP (forskolin and 8-bromoadenosine cyclic-3',5'-monophosphate) levels were found to mimic the inhibitory action of MO-IH on MF synthesis. To further support these findings, the effect of MO-IH-1 on production of cAMP was investigated. The results demonstrated that MO-IH stimulated a dose-dependent increase in cAMP levels. Furthermore, a maximal 2-fold increase in cAMP was detected after a 5-min exposure of MO membranes to 100 nM MO-IH-1, falling to basal levels thereafter. The results presented strongly support a role for cAMP in the signal transduction mechanism of MO-IH that leads to inhibition of MF synthesis in MOs.

Phytoecdysteroids in seeds and plants of Rhagodia baccata (Labill.) Moq. (Chenopodiaceae).

Seeds of Rhagodia baccata afforded, in addition to 20-hydroxy-ecdysone and polypodine B, a novel phytoecdysteroid, (20R)-22-deoxy-20,21-dihydroxyecdysone, the structure of which was elucidated unequivocally by UV, LSIMS and NMR techniques. This compound possessed agonistic activity in the Drosophila melanogaster BII cell bioassay, with an ED50 value of 2.0 x 10(-7)M (ED50 value for 20-hydroxyecdysone = 7.5 x 10(-9)M). The distribution of ecdysteroids in plants of R. baccata has been determined. Highest levels are associated with the youngest aerial tissues and with the roots. Ecdysteroid profiles are qualitatively very similar throughout the plant, with 20-hydroxyecdysone and polypodine B predominating in all plant parts tested.

Molecular cloning and characterization of hemolymph 3-dehydroecdysone 3beta-reductase from the cotton leafworm, Spodoptera littoralis. A new member of the third superfamily of oxidoreductases.

The primary product of the prothoracic glands of last instar larvae of Spodoptera littoralis is 3-dehydroecdysone (3DE). After secretion, 3DE is reduced to ecdysone by 3DE 3beta-reductase in the hemolymph. We have previously purified and characterized 3DE 3beta-reductase from the hemolymph of S. littoralis. In this study, cDNA clones encoding the enzyme were obtained by reverse transcription-polymerase chain reaction, employing primers based on the amino acid sequences, in conjunction with 5'- and 3'-rapid amplification of cDNA ends. Multiple polyadenylation signals and AT-rich elements were found in the 3'-untranslated region, suggesting that this region may have a role in regulation of expression of the gene. Conceptual translation and amino acid sequence analysis suggest that 3DE 3beta-reductase from S. littoralis is a new member of the third superfamily of oxidoreductases. Northern analysis shows that 3DE 3beta-reductase mRNA transcripts are widely distributed, but are differentially expressed, in some tissues. The developmental profile of the mRNA revealed that the gene encoding 3DE 3beta-reductase is only transcribed in the second half of the last larval instar and that this fluctuation in expression accounts for the change in the enzyme activity during the instar. Southern analysis indicates that the 3DE 3beta-reductase is encoded by a single gene, which probably contains at least one intron.

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Neuropeptide regulation of biosynthesis of the juvenoid, methyl farnesoate, in the edible crab, Cancer pagurus.

The neuropeptide mandibular organ (MO)-inhibiting hormone (MO-IH), synthesized and secreted from the X-organ-sinus-gland complex of the eyestalk, regulates the biosynthesis of the putative crustacean juvenile hormone, methyl farnesoate (MF). Using radiolabelled acetate as a precursor for isoprenoid biosynthesis, farnesoic acid (FA), farnesol, farnesal, MF and geranyl geraniol were detected in MOs cultured for 24 h. Treatment of MOs with extract of sinus gland inhibited the final step of biosynthesis of MF, catalysed by FA O-methyltransferase. Additionally, treatment of MOs with purified MO-IH exhibited a dose-dependent inhibition of this final step of MF synthesis. The extent of this inhibition was dependent on the ovary stage of the MO-donor animal, being maximal in MOs from animals in the early stages of ovarian development. Assay of FA O-methyltransferase activity, using [3H]FA in the presence of S-adenosyl-l-methionine, demonstrated that the enzyme was located in the cytosolic fraction of MOs and was inhibited by incubation of MOs with MO-IH prior to preparation of subcellular fractions. For cytosolic preparations taken from vitellogenic animals, both Vmax and Km were appreciably lower than for those taken from non-vitellogenic animals. Conversely, eyestalk ablation of early-vitellogenic animals, which removes the source of MO-IH in vivo, resulted in enhancement of the cytosolic FA O-methyltransferase activity. Although both Vmax and Km show an appreciable increase upon eyestalk ablation, the increased enzyme activity is probably reflected by the fact that Vmax/Km (an approximate indication of kcat) has increased 5-fold. The combined evidence demonstrates that MO-IH inhibits FA O-methyltransferase, the enzyme which catalyses the final step of MF biosynthesis in MOs.

Isoprenylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of isoprenyl groups, is a widespread phenomenon. Here we provide direct evidence for this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of [3H]mevalonolactone, specific C. elegans polypeptides became labelled in both aqueous and detergent (Triton X-114)-enriched extracts. Chemical and GC-MS analysis of modifying groups, cleaved from C. elegans polypeptides, revealed that geranylgeranylation and, to a lesser extent, farnesylation of target polypeptides occurred. Immunoblot analysis provided preliminary evidence that the ras-like let-60 polypeptide was a target for isoprenylation in C. elegans.

Fatty acylation of polypeptides in the nematode Caenorhabditis elegans.

Covalent modification of eucaryotic proteins, involving addition of fatty acyl groups, is a widespread phenomenon. Here we describe the occurrence of this form of covalent modification in the free-living nematode, Caenorhabditis elegans. Following incubation in the presence of either [3H]-myristic acid or [3H]-palmitic acid, specific C. elegans polypeptides became labelled. Chemical analysis revealed that following incubation of C. elegans with [3H]-myristic acid, polypeptides became labelled with myristoyl, palmitoyl or stearoyl moieties; after incubation with [3H]-palmitic acid, palmitoyl or stearoyl moieties were incorporated into polypeptides. Fatty acyl groups were linked to target polypeptides, predominantly through alkali-labile thioester or ester linkages and acid-labile amide linkages. Where myristoylation involved an amide linkage, the modified amino acid was usually glycine. Preliminary immunological evidence indicated that heterotrimeric GTP-binding protein alpha subunit(s) are possible target(s) for acylation in C. elegans.

1 alpha,20R-dihydroxyecdysone from Axyris amaranthoides.

Bioassay/RIA-directed phytochemical examination of the seeds of Axyris amaranthoides afforded a new ecdysteroid: 1 alpha,20R-dihydroxyecdysone [1-epi-integristerone A], together with 20-hydroxyecdysone and polypodine B. The structure of 1 alpha,20R-dihydroxyecdysone was determined unequivocally by UV, LSIMS, and a combination of 1D and 2D NMR techniques.

N-Myristoylation of the catalytic subunit of cAMP-dependent protein kinase in the free-living nematode Caenorhabditis elegans.

N-Myristoylation of the catalytic subunit (C-subunit) of cAMP-dependent protein kinase is widespread in animal cells. Some invertebrates express non-myristoylated isoforms of C-subunit but these co-exist with at least one myristoylated isoform. The generality of this observation implies an indispensable function for myristoylated C-subunit, but notwithstanding this, neither of the C-subunit isoforms hitherto described in C. elegans is apparently N-myristoylated. In light of this anomaly, the myristoylation status of the C-subunit has been examined in adult C. elegans. Evidence is presented for the presence of an N-myristoylated isoform.