Juvenile Hormone Binding Research Articles

Identification, characterization, and developmental profile of a high molecular weight, juvenile hormone‐binding protein in the hemolymph of the migratory grasshopper, Melanoplus sanguinipes

AbstractIn the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a Mr of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a Mr 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS‐treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley‐Liss, Inc.

Juvenile hormone binding components of locust fat body

AbstractJuvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[3H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [3H]methoprene and [3H]pyriproxyfen, on the other hand, were obscured by abundant non‐specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000‐fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley‐Liss, Inc.

Juvenile hormone regulation of hemolymph juvenile hormone binding protein in the black strain of the tobacco hornworm, Manduca sexta

AbstractNumerous studies have demonstrated regulation of specific lepidopteran proteins by pharmacological doses of insect juvenile hormone (JH). In this study, topical application of a 1 pg dose of JH I to fourth stadium larvae of the black (bl) mutant strain of the tobacco hornworm, Manduca sexta, induced a 50% increase in the titer of hemolymph juvenile hormone binding protein (hJHBP). Radioimmunoassay confirmed that JH titers were lower in bl larvae than in wild‐type larvae at the time of JH treatment. Enzyme immunoassay analysis of hJHBP titers demonstrated that regulation by JH I was dose‐dependent at doses up to 10 pg and that the response was saturated above 100 pg. Western blotting and equilibrium dialysis confirmed these results and demonstrated that hJHBP from bl larvae had the same molecular mass and displayed the same affinity for JH I as hJHBP isolated from wild‐type larvae. Time course studies showed that regulation was complex: 1 2 h after JH I treatment, hJHBP titers were twofold lower in treated than in control bl larvae, while 44 h after treatment they were twofold higher. JH I regulation of hJHBP titers in bl larvae was independent of changes in total hemolymph protein. © 1995 Wiley‐Liss, Inc.

JUVENILE HORMONE BINDING PROTEIN IN THE OVARIES OF HOUSEFLY MUSCA DOMESTICA VACINA

Abstract By using charcocal binding assay, the juvenile hormone binding protein (JHBP) was determined in the ovaries of houseflies. This ovarian JHBP possesses high affinity with juvenile hormone III (JH III) and has a Kd of 2.1 III 10‐‐8 M. The binding of 3H‐juvenile hormone III (3H‐JH III) to this protein was inhibited by unlablled JH III, but not by juvenile hormone analog ZR 512 or ZR 515. The level of this ovarian JHBP reached the highest in houseflies 48 h after emergence, and was 6. 5‐fold and 15. 5‐fold higher than that in housefIies 60 h and 72 h after emergence, respectively. No binding activity was detected in the ovaries of houseflies 24 h or 36 h after emergence. The absence of JHBP in the ovaries of houseflies 36 h after emergence could be reversed by applying JH III to newly emerged houseflies. The data suggest that the fluctuation of the JHBP concentration might associate with the action of juvenile hormone (JH) on housefly vitellogenesis.

Characterization of a juvenile hormone binding lipophorin from the blowfly Lucilia cuprina

The larval haemolymph of the sheep blowfly Lucilia cuprina (Weidemann) contains a juvenile hormone binding protein with a K d for racemic JH III of 33 ± 6 nM. The density of the binding sites is 212 ± 33 pmol/mg haemolymph protein. The binding protein is equally specific for JH III and methyl farnesoate. Some natural juvenoids were ranked for their ability to displace [ 3H]JH III with JH III > JH II > JH I > JH III > JH III diol > JHB 3 = no detectable displacement. These data, together with displacement studies for 14 synthetic juvenoids, indicate some characteristics of the JH binding cleft. The binding protein is a high density lipophorin (density = 1.15 g/ml) and has subunit molecular weights of 228 kDa (apolipophorin I) and 70 kDa (apolipophorin II). The N-terminal amino acid sequences of the subunits have no discernible homology to any previously sequenced protein. Lipophorin-specific immunocytochemical staining occurs in a subset of fat body cells.

Larva lights: A decade of photoaffinity labeling with juvenile hormone analogues

The introduction of photoaffinity labeling into the mode of action of insect hormones and pheromones started 12 yr ago with the photoaffinity labeling of juvenile hormone binding proteins (JHBPs) from cockroaches in the laboratory of the late John K. Koeppe. Applying this technique to Manduca sexta led ultimately to a three-laboratory collaborative project that has begun to dissect the molecular basis for JH transport, metabolism, and nuclear binding and gene activation in Lepidoptera. This review provides (1) a history of the first experiments; (2) an idea of the breadth of the technique in the arthropod classes Insecta, Crustacea, and Arachnida; and (3) evidence for the depth of the technique in unearthing key details about three different types of the molecular action of JH in M. sexta.

Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm, Bombyx mori

A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 × 10 −8 M for JH I, 1.14 × 10 −7 M for JH II and 3.9 × 10 −7 M for JH III, and thus its affinity for JH analogues is in the order of JH I > JH II > JH III. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.

Juvenile hormone and hemolymph juvenile hormone binding protein titers and their interaction in the hemolymph of fourth stadium Manduca sexta

A driving force in the initiation of an endocrine response is the modulation of hormone titers surrounding the responsive tissue. For nonpolar hormones, titer fluctuations are, in part, regulated by specific circulatory transport proteins that bind, protect and convey the hormones to the responsive tissues. In the present study, juvenile hormone (JH), hemolymph juvenile hormone binding protein (hJHBP), and total hemolymph protein in the fourth larval stadium of Manduca sexta were quantified from similarly staged insects to assess whether JH is bound or unbound when it interacts with the target. JH levels, as determined by radioimmunoassay, were highest, 12.87 nM, 4 h after ecdysis to the fourth stadium, and declined 6-fold to the lowest titer, 2.28 nM, at 44 h after ecydsis, i.e. 8 h after head critical period. Conversely, hJHBP titers peaked at 11.95 μM 44 h after ecdysis and dropped 5-fold by the time of ecdysis to the fifth stadium. Total hemolymph protein also reached its highest level, 19.83 mg/ml, at 44 h; however, hJHBP titers did not follow fluctuations in the total hemolymph protein. Calculations of bound and unbound JH indicated that >99% of the hormone was bound by hJHBP. In contrast, <1% of the binding protein was hormone-loaded at any time during the stadium. Due to the rapid metabolism of JH in the hemol the hemolymph is improbable at certain times during development. Thus, the rate limiting step in JH uptake at these times may be the interaction of hJHBP with the target cell.

Role of juvenile hormone binding protein in modulating function of JH epoxide hydrolase in eggs of Manduca sexta

Juvenile hormone (JH) is catabolized primarily by two enzymes, JH esterase (JHE) and JH epoxide hydrolase (JHEH). A JH binding protein (JHBP) may modulate this degradation by ligand sequestration. In this study, the effects of JHBP on the metabolism of JH by JHEH were examined. 1. (1) Purified JHEH from Manduca sexta (Lepidoptera) eggs converted JH to JH-diol four to five times faster than JH-acid was converted to JH-acid-diol in the absence of JHBP. 2. (2) The presence of JHBP dramatically decreased the degradation of JH, but not that of JH-acid. Similar results were obtained by using crude unsolubilized microsomes. 3. (3) The inhibition of JHEH activity by JHBP was due to the formation of a JH-JHBP complex that could be detected at the origin of a silica gel TLC plate. 4. (4) A crude homogenate of newly oviposited M. sexta eggs was incubated with a physiological concentration of JH. JH-acid-diol and JH-acid were found as the major metabolites, and the rate of hydration of JH-acid by JHEH was faster than that of JH. These results suggested that JH was protected from JHEH by cytosolic JHBP as a JH-JHBP complex; in contrast, JH-acid, a poor ligand for JHBP, was efficiently metabolized into JH-acid-diol by JHEH in vivo. Thus, while JH was not efficiently degraded by JHEH in the presence of JHBP, JHEH could nevertheless act as the ultimate scavenger for JH by hydrating JH-acid generated by JHE during embryogenesis in vivo.

Photoaffinity labeling of juvenile hormone epoxide hydrolase and JH-binding proteins during ovarian and egg development in Manduca sexta

Developmental profiles of a membrane-associated 50 kDa juvenile hormone epoxide hydrolase (JHEH) and a cytosolic 32 kDa juvenile hormone binding protein (JHBP) in the ovaries and eggs of Manduca sexta were obtained using photoaffinity analogs of JHs. The 50 kDa JHEH was present at a constant level in adult female ovaries, in eggs, and in first instar larvae. In contrast, the cytosolic 32 kDa JHBP was absent in pupae and increased significantly in the adult. The cytosolic 32 kDa JHBP in eggs had properties similar to the hemolymph 32 kDa JHBP in terms of size, isoelectric point, ligand specificity, and immunoreactivity. The level of hemolymph 32 kDa JHBP, however, was present throughout pupal and adult stages. The JH-specific esterase (JHE) activity in ovaries also increased in the adult, remained unchanged until oviposition, and decreased in eggs between days 1 and 2 after oviposition. These results show an increase of cytosolic JHBP and JHE at the termination phase of vitellogenesis and oogenesis. Moreover, it appears that high JHE and JHEH activity in the embryo helps maintain a sufficiently low JH titer to circumvent inhibition of embryonic development. Finally, a hemolymph 80 kDa protein, which was present in larvae and pupae but was absent in the newly eclosed adult female, was also specifically labeled by the JH photoaffinity analogs.

Developmental profiles and characteristics of hemolymph juvenile hormone esterase, general esterase and juvenile hormone binding in the cricket, Gryllus assimilis

Developmental profiles of serum juvenile hormone esterase (JHE) and general esterase (GE) during the last stadium of the cricket Gryllus assimilis were highly concordant and exhibited a mid-stadium peak; juvenile hormone binding activity (JHB) increased continuously throughout the stadium. JHE activities were the highest yet reported in the Orthoptera (peak = 130 nmol/min/ml serum), the enzyme was inhibited strongly by trifluoropropanones ( I 50 = 10 −8−10 −9) but only weakly by DFP (10 −6) or eserine (10 −4) and K m(JH-III) was (16–3 nM). Inhibition studies indicated that the majority of GE activity was due to an enzyme different from JHE. These data provide the background for a study of the microevolution of the endocrine system in G. assimilis.

Covalent modificaition of juvenile hormone binding proteins by photoaffinity labeling: An unexpected gel shift effect

The 32 kD juvenile hormone binding protein (JHBP) and two 80 kD proteins in larval Manduca sexta hemolymph were labeled with [3H]FDK, a photoaffinity analog of methyl farnesoate (MF). The labeling could be completely displaced by a 30-fold excess of either MF or JH II, demonstrating that [3H]FDK binds specifically to the JH binding sites of the 32 kD JHBP and the 80 kD proteins. In addition, a high molecular-mass protein was labeled with [3H]FDK; labeling could be displaced by excess MF but not by JH II, demonstrating the selectivity in binding MF. The 32 kD JHBP also appeared to weakly bind the potent juvenoid, methoprene, at the JH binding site. Covalent modification by [3H]FDK induced a change in the apparent size and the isoelectric point of the JHBP. These changes were not induced by substrate alone, nor by UV irradiation alone. The same effect was also observed during labeling with [3H]MDK, an analog of methoprene. These data provide an important caveat for anticipating artifactual changes of protein properties during chemical or photochemical affinity labeling experiments. The molecular dimensions of [3H]FDK more closely resemble those of JH II than those of [3H]EHDA, a photoactivatable analog of JH II. We suggest that covalent modification by a diazoketone photolabel involves a hydrophilic amino acid important in the recognition of the ester group of JH. © 1994 Wiley-Liss, Inc.

Benzodioxole‐1,3‐benzodioxole derivatives and their effects on the reproductive physiology of insects

AbstractAlkyl ethers of methylenedioxy analogs of obtusastyrene or benzyl‐1,3‐benzodioxole derivatives (BBDs) and related benzylphenols have been shown to interfere with various phases of reproduction in insects. BBDs have also been shown to interfere with sex attractancy, to induce precocious development, and to antagonize juvenile hormone (JH) functions in insects. Because representative BBDs were reported to show low toxicity to mammals and to be negative in assays testing for potential mutagens, these compounds held much promise to be environmentally safe insect chemosterilants. The mode of action of BBDs does not involve blocking or competition for putative JH receptor sites on follicular cells or hemolymph JH binding proteins. However, BBDs were shown to interfere with (1) in vitro biosynthesis and release of JH from corpora allata of Mediterranean fruit fly females, and (2) microtubule assembly in insects. © 1994 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Juvenile hormone epoxide hydrolase. Photoaffinity labeling, purification, and characterization from tobacco hornworm eggs.

Juvenile hormone epoxide hydrolase (JHEH), which may play a pivotal role in regulating insect juvenile hormone (JH) titer along with JH esterase, was identified in tobacco hornworm (Manduca sexta) eggs by using photoaffinity analogs of JHs. The UV light-induced covalent labeling with [3H]epoxyhomofarnesyl diazoacetate, a JHII analog, revealed a membrane-associated 50-kDa protein that was selectively and specifically labeled. This 50-kDa protein was copurified 171-fold with the JHEH activity to homogeneity through DEAE-Sephacel, Mono Q, and hydroxylapatite columns, which led us to conclude that the labeled 50-kDa protein was a JHEH. The steady-state kinetics of the purified microsomal JHEH showed that it followed Michaelis-Menten kinetics with Km values of 0.61, 0.55, and 0.28 microM for JHI, II, and III, respectively, and that JHIII showed a significantly higher Vmax than JHI or JHII. JH acid was also converted to the corresponding diol at a rate 4-fold slower than the corresponding JH. Thus, the differences in the binding of substrate and the rate of turnover by JHEH were affected by the epoxyfarnesoate ester moiety of JH and the difference between the cis-11-methyl group of JHIII versus the cis-11-ethyl group of JHI and II. Purified JHEH showed optimal enzyme activity at pH 7.5-8.5. Interestingly, the presence of recombinant M. sexta JH binding protein (JHBP) dramatically decreased the degradation of JH by JHEH in vitro. Since the cytosolic JHBP in eggs closely resembles the hemolymph JHBP, we suggest that cytosolic JHBP may play a role in protecting JHs from JHEH in vivo. Furthermore, JHEH may play a significant role in the secondary metabolism of JH acid generated by JH esterase.

Affinity purification and binding analysis of the hemolymph juvenile hormone binding protein from Manduca sexta.

A high-affinity juvenile hormone binding protein was purified from the hemolymph of the tobacco hornworm, Manduca sexta, employing ammonium sulfate precipitation and affinity and size-separation chromatography. The naturally occurring enantiomer of juvenile hormone III (10R) was converted to juvenile hormone III acid and then covalently attached to aminohexyl-Sepharose 4B. Hemolymph from early fifth stadium (60 h postecdysis) larvae was used as the source of hJHBP. The yield of hJHBP was approximately 25% of the starting material, with 3.5 mg of highly purified, biologically active hJHBP recovered from 100 mL of hemolymph. Binding parameters were examined using equilibrium dialysis and highly purified, enantiomerically correct juvenile hormone I and II and racemic JH III. The equilibrium dissociation constants for juvenile hormone I and II were approximately 6 x 10(-10) M at 4 degrees C, while racemic juvenile hormone III displayed an equilibrium dissociation constant of 1.9 x 10(-9) M. At 25 degrees C the equilibrium dissociation constant for juvenile hormone I was 1.6 x 10(-9) M. Half-times of dissociation were also determined for the three homologs. The half-time of dissociation was 30 s for juvenile hormone I, 20 s for juvenile hormone II, and 13 s for juvenile hormone III at either 4 or 25 degrees C. Using the new equilibrium dissociation constants, we calculate that better than 99% of the circulating juvenile hormone titer may be bound to this hemolymph protein.

Juvenile hormone binding proteins in larval fat body nuclei of Drosophila melanogaster

In previous studies juvenile hormone has been implicated as acting at the plasma membrane level or at the chromosomal level. The present study has examined juvenile hormone III binding and action in the nucleus of two Drosophila melanogaster tissues that respond to juvenile hormone. Juvenile hormone III was found to bind to nuclear preparations of larval fat body, a tissue that in adults undergoes histolysis in response to juvenile hormone. Photoaffinity labeling revealed the presence of three nuclear juvenile hormone analogue-binding proteins, two of which can be specifically competed with juvenile hormone III. One of these proteins corresponds in size to a juvenile hormone binding protein found in greater quantity in the cytosolic fraction from several tissues of Drosophila, and the other protein is unique to the nuclear fraction. Juvenile hormone III acquired the ability to bind to DNA-cellulose when incubated with larval fat body cytosol, but not with larval hemolymph. Moreover, juvenile hormone III was shown to stimulate RNA synthesis in male accessory glands, a tissue that responds to juvenile hormone by increased protein synthesis in several insect species. These results suggest that the juvenile hormone may act through the nucleus to effect at least part of its action.

Analysis and Modification of Thiols in the Hemolymph Juvenile Hormone Binding Protein of Manduca sexta

Hemolymph-induced in vitro modifications of the hemolymph juvenile hormone binding protein from the tobacco hornworm, Manduca sexta, were analyzed by polyacrylamide gel electrophoresis, Western blots, and equilibrium dialysis. Upon hemolymph melanization, a complex reaction involving polymerization of phenolic compounds, multiple forms of the protein were detected. Under melanizing conditions, one slower- and several faster-migrating proteins were observed. When the hemolymph was treated with 100 mM catechol, a melanin precursor, two well-defined forms of the binding protein appeared. When the protein was incubated with excess catechol (>100 mM) a single faster-migrating form appeared. Both fast and slow forms bound juvenile hormone I with similar efficiency. These results were duplicated using the thiol modifying reagents p-chloromercurobenzoate and N-ethylmaleimide, suggesting modification of cysteine residues. Using differential alkylation, it was determined that hemolymph juvenile hormone binding protein contained two cystine and two cysteine residues. One of the cysteines is exposed and readily accessible for modification. Since modification of the exposed free thiol did not alter binding, it presumably resides outside the hormone-binding domain. The analyses support the observation that there is a single form of the hemolymph juvenile hormone-binding protein in our strain of M.sexta.

Ligand binding by a recombinant insect juvenile hormone binding protein.

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been isolated, sequenced, and expressed in an insect cell line. A recombinant baculovirus, containing the JHBP cDNA fused to the p10 promoter of Autographa californica nuclear polyhedrosis virus, was constructed. Insect cells (Sf9) infected with this virus secreted recombinant JHBP (rJHBP) into the medium (> 50 micrograms/mL), and cotranslational removal of an 18 amino acid leader sequence was observed. rJHBP was cross-reactive with an antiserum prepared to the hemolymph JHBP and was specifically labeled by [3H]EHDA, a photoaffinity analog of JH II, demonstrating that rJHBP was an isoform of the previously reported 32-kDa JHBP [Lerro, K. A., & Prestwich, G.D. (1990) J. Biol. Chem. 265, 19800-19806]. rJHBP was purified from insect cell medium to homogeneity by ion-exchange and gel-filtration chromatography. The purified rJHBP had a higher affinity (KD = 11 nM for JH I and KD = 42 nM for JH II) than that reported for crude hemolymph JHBP (KD = 80 nM for JH I). The circular dichroism (CD) spectrum of purified rJHBP indicated 34% alpha-helix and 23% beta-sheet. The CD spectra of rJHBP in the presence and absence of JH II were the same, indicating no change in secondary structure induced by ligand binding. Thus, the rJHBP expressed in insect cells binds JHs and is suitable for structural and functional analysis.

Changes in the titre of a juvenile hormone III binding lipophorin in the haemolymph of Diploptera punctata during development and reproduction: Functional significance

A monoclonal antibody specific to juvenile hormone binding lipophorin was used in an enzyme-linked immunosorbent assay (ELISA) to determine titre of this protein in the haemolymph during the fourth stadium and the first reproductive cycle of adult female Diploptera punctata. Juvenile hormone binding lipophorin was present in the haemolymph at all times at concentrations between 3.75 and 16 μg/μl and its titre exhibited some changes which were not coincident with those in total haemolymph protein concentration. Three aspects of juvenile hormone binding lipophorin function were examined: (1) Plasma juvenile hormone esterase activity and partially purified juvenile hormone esterase activity was inhibited by exogenous juvenile hormone binding lipophorin in a dose-dependent manner by up to 92%. 10 R-juvenile hormone III was metabolized faster by diluted plasma than racemic juvenile hormone III, both in the presence and absence of exogenous juvenile hormone binding lipophorin. (2) Exogenous juvenile hormone binding lipophorin did not affect the biosynthesis and release of juvenile hormone from the corpora allata of day-2 virgin females in vitro. (3) The amount of juvenile hormone binding lipophorin-immunoreactive material in the ovaries of mated females was determined by ELISA. Juvenile hormone binding lipophorin-immunoreactive material reached a maximum in day-3 mated female ovaries (23 μg/pair) at the start of vitellogenesis and declined rapidly thereafter.

The structure of a juvenile hormone-binding lipophorin from the hemolymph of Diploptera punctata

The high molecular weight, high affinity juvenile hormone binding protein from the hemolymph of Diploptera punctata was identified as a lipophorin by gradient KBr ultracentrifugation and SDS gradient PAGE. This juvenile hormone binding lipophorin (JHBL) was composed of two subunits, apolipoprotein I (230 kDa mol. wt) and apolipoprotein II (80 kDa mol. wt). The density of the native protein was 1.15 g/ml. Photoaffinity labeling using the JH analog [ 3H]EFDA demonstrated that the JH binding site resides on apolipoprotein I. The amino acid composition of both native lipophorin and its two subunits was determined and the N-terminal sequence of the 80 kDa apolipoprotein described for 19 of the first 21 amino acids. This sequence did not have similarity to any known protein. The N-terminus of the 230 kDa apolipoprotein was blocked. The specificity of a monoclonal antibody to purified native JHBL was also demonstrated. We show that the monoclonal antibody was specific to the 230 kDa subunit and did not recognize the 80 kDa apolipoprotein.