Enzyme-amplified enzyme-linked immunosorbent assay for gibberellin A4 based on the NAD-dependent redox cycle

An antiserum against gibberellin A₄ (GA₄) raised in rabbits and its partially purified antibodies were used to develop radioimmunoassay (RIA) and indirect enzyme-linked immunosorbent assays (ELISAs) for GA₄. Of three immunoassays tested, an ELISA based on the NAD-dependent redox cycle (enzyme-amplified ELISA) had highest sensitivity. Levels of methylated GA₄ detected by this most sensitive method ranged from 0.1 fmol/assay (3.5 fg/assay) to 0.1 pmol/assay (3.5 pg/assay) suggesting applicability of this method to the detection of gibberellins in purified plant extracts.     

A chemical approach to solving bridging phenomena in steroid radioimmunoassays

Steroid radioimmunoassays (RIA) employ antibodies raised against a carrier protein-steroid conjugate. Individual antibodies may recognize the steroid, the protein or the chemical bridge used to Join them together. Use of the same bridge In the tracer results in higher affinity binding of the tracer than the native ligand which in turn results in a loss of sensitivity and precision. We have greatly reduced bridge-binding In a RIA for androstenedione. Conjugates and radioiodinated labels were prepared with either an ester op ether chemical bridge. By using an antibody and the corresponding label with the heterologous bridge very sensitive assays were obtained.     

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.     

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.     

Radioreceptor assay for atrial natriuretic factor

Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

An enzyme-linked immunosorbent assay (ELISA) for plasma testosterone

A rapid, single extraction ELISA for testosterone in plasma is described, using a standard 96 well microtitre plate. Testosterone is covalently bonded to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to the ELISA plate, giving an immobilised antigen approach which simplifies subsequent assay standardisation for steroid hormone assays. The addition of standard, sample and first antibody (rabbit anti-testosterone), which is unique for each different assay, is followed by a general procedure which includes washing, addition of peroxidase labelled goat antirabbit IgG, further washing and finally, addition of o-phenylenediamine substrate with colour development and reading of the plate at 492 nm on an automatic ELISA processor. The ELISA assay is compared to a testosterone RIA with 125I-label and has similar specificity and precision to the latter with a quicker processing time, and is more cost effective. The added advantages that ELISA assays confer over RIAs in terms of isotope purchase and disposal make this an ideal procedure for use in a routine steroid laboratory.     

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.     

Preparation and Validation of an Antiserum Specific for Non-Derivatized Gibberellins

An antiserum recognizing free gibberellins (GAs) was preparedby immunizing rabbits with a GA₄-BSA conjugate. A radioimmunoassay(RIA) and an enzyme-linked immunosorbent assay (ELISA) wereset up using this antiserum. This antiserum showed high cross-reactivityto the so-called active GAs, such as GA¹, GA₃, GA⁴ and GA₇.The range for measurements of these gibberellins extended from30 fmol to 3 pmol in both RIA and ELISA. Extracts from immature seeds of P. vulgaris were subjected todetermination of GA, by RIA and GC/SIM. The two assays providedsimilar results, indicating the high degree of reliability ofthe immunoassay. ²Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Mine-machi 350, Utsunomiya, 321 Japan ( Accepted August 8, 1990)     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.     

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.     

A competitive enzyme-linked immunosorbent assay for plasma 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione)

An enzyme-linked immunosorbent assay (ELISA) for 11-deoxycortisol is described for the first time. 11-Deoxycortisol-thyroglobulin conjugate is adsorbed onto the wells of a 96-well ELISA plate and competes with 11-deoxycortisol in the standards or plasma extract for antibody binding sites. After washing, immobilized primary antibody is probed with peroxidase-labeled goat anti-rabbit IgG. The ELISA plate is further washed and o-phenylenediamine added, color developed and the absorbance read at 492 nm. The ELISA shows good agreement with our existing 11-deoxycortisol radioimmunoassay (RIA) and has similar specificity and performance which allow its use in the routine steroid laboratory for assessing pituitary adrenal function by the metyrapone test.     

Development of an in vitro assay for prothoracicotropic hormone of the gypsy moth,Lymantria dispar (L.) following studies on identification,titers and synthesis of ecdysteroids in last-instar females

Summary Hemolymph ecdysteroid titers and in vitro prothoracic gland ecdysteroid synthesis have been examined in last-instar larval (5th instar) females of Lymantria dispar. Ecdysteroids were quantified by radioimmunoassay and characterized by co-elution with known standards of ecdysteroids on reverse-phase high-performance liquid chromatography. Analysis of hemolymph yielded ecdysone and 20-OH-ecdysone in ratios of 1:1 (day 6, shortly after attainment of maximum weight) and 1:28 (day 10, molting peak). Analysis of in vitro culture media from glands challenged with extracts of brains or retrocerebral complexes, or left unchallenged, revealed only immunoreactive material co-eluting with a known standard of ecdysone. Time-course studies of in vitro prothoracic gland ecdysone secretion demonstrated a major peak on day 10, 1–2 days prior to pupal ecdysis, and a small elevation on days 5–6. On days 5 and 6, 2.29±0.41 and 2.65±0.72 ng ecdysone per gland, respectively, were secreted in 6-h cultures. On day 10, 25.69±4.36 ng was secreted in 6-h culture. The ability of prothoracic glands of various ages to respond to brain extracts containing prothoracicotropic hormone activity was tested by determining an activation ratio for each day of the instar. The activation ratio was determined over a 90-min period by dividing the amount of ecdysone secreted by one member of a pair of prothoracic glands in the presence of brain extract by that of its contralateral control gland in Grace's medium. Prior to the addition of brain extract, the activity of the glands was allowed to subside to basal level for 180 min in Grace's medium. The activition ratio was highest on days 3–7 and fell throughout the remainder of the instar as the inherent ability of the prothoracic gland to maintain high levels of ecdysteroid synthesis in vitro in the absence of prothoracicotropic hormone increased. A two-phase in vitro assay for prothoracicotropic hormone was established using activition ratios. This assay showed saturable doseresponse kinetics for prothoracic gland ecdysone secretion and specificity to extracts prepared from brain or retrocerebral complexes. A comparable assay for prothoracicotropic hormone purification, based on net synthesis and requiring half the number of prothoracic glands was also established.Abbreviations A _r activation ratio - HPLC high performance liquid chromatography - HPSEC high performance size-exclusion chromatography - PG prothoracic gland - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.

We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.     

Assessment of delta-opioid receptor activation by a series of peptides in cultured cells.

This study was designed to investigate the relative ability of a series of cyclic opioid peptides to initiate the first activation steps following their binding of delta-opioid receptors. The extent of stimulation of low Km guanosine-triphosphatase (GTPase activity) and inhibition of hormonally-stimulated cAMP accumulation in the NG108-15 (neuroblastoma-glioma) hybrid cell line were determined and compared for six closely related peptides. In addition, their binding affinity was assessed by competition with 3H-[D-Pen2D-Pen5]-enkephalin (3H-DPDPE) in membranes from these cells. All peptides tested elicited comparable maximal effects for both functional responses. Different potencies in stimulating the low Km GTPase was observed at sub-maximal agonist concentrations, although the shallow dose-response behavior did not allow accurate determination of ED50s. Estimation of ED50s for inhibition of cAMP accumulation could be made by curve fitting and were similar for four of these peptides, while DCDPE and 3R-methylDCDPE, the highest affinity analogs, were considerably more potent. In general, the observed differences in hormonal activity somewhat parallel the rank order of binding affinities, but no strict relationship was found between receptor binding and activation.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: effects of the ecdysone agonist RH 5849 on ecdysone 20-monooxygenase activity

The non-steroidal ecdysone agonist RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) was found to inhibit in a dose-response and apparently competitive fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity in the midgut of wandering stage last instar larvae of the tobacco hornworn, Manduca sexta. More effectively on a per molar basis than the naturally occurring molting hormones ecdysone and 20-hydroxyecdysone, RH 5849 was also found to elicit the dramatic 50-fold increase in midgut steroid hydroxylase activity (which normally occurs with the onset of the wandering stage) when injected into competent head or thoracic ligated pre-wandering last instar larvae. These data support and extend the potential usefulness of RH 5849 as a pharmacological probe for further investigating the actions of ecdysteroids and their role(s) in the regulation of ecdysteroid monooxygenases.     

Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.     

Detection of Synenkephalin, the Amino-Terminal Portion of Proenkephalin, by Antisera Directed Against Its Carboxyl Terminus

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.     

Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta

The metabolism and distribution of endogenous ecdysone and injected [³H]ecdysone were studied during the pupal-adult development of Manduca sexta. Well-characterized antisera were used to detect and quantify endogenous metabolites by radioimmunoassay (RIA) following their separation by ion-suppressed reverse phase, and normal phase, high performance liquid chromatography. Identical chromatographic procedures were employed to determine the metabolic fate of the [³H]ecdysone in the haemolymph pool. These studies revealed the sequential appearance in the haemolymph and gut of progressively oxidized metabolites of ecdysone—hydroxylation at C-20 was followed by hydroxylation at C-26. The data are suggestive of both the induction of the steroid hydroxylases (oxidases) by substrate or other effector substances and the possible coordination of developmental events by ecdysteroids other than 20-hydroxyecdysone.In the haemolymph, two highly-polar conjugates of ecdysone were observed together with conjugates of the other free ecdysteroids, especially those hydroxylated at C-26. In contrast, relatively little 20-hydroxycdysone conjugate was detected in the insect. As adult development proceeded, both endogenous and radiolabelled ecdysteroids were increasingly localized in the gut, so that just prior to eclosion most ecdysteroids were present in the meconium of the high gut (rectal pouch). The peak titres and the kinetics of appearance of ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were similar for both haemolymph and gut (and for males and females), but considerably higher levels of C-26 oxidized (acid) metabolites of ecdysone and 20-hydroxyecdysone were localized in the gut. Although levels of highly-polar ecdysteroid conjugates found in the haemolymph and gut were similar, considerable amounts of three less polar ecdysone conjugates, of 3-α-epimers of ecdysone and 20-hydroxyecdysone, and of a substance tentatively identified as 2-deoxyecdysone were found only in the gut. Whether ionized, conjugated, or free, the gut ecdysteroids did not appear to equilibrate with the haemolymph compartment.Differences were observed in the metabolism kinetics of exogenously administered radiolabelled ecdysone when compared to the endogenous ecdysteroids; and some RIA positive gut metabolites did not become significantly radiolabelled. This suggests that injection of ecdysone may not simulate the endogenous secretion of ecdysone or its subsequent metabolism and distribution completely accurately.