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 JHI >JHII >JHIII. 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-.     

Synthesis and binding affinity of an iodinated juvenile hormone

The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural 3H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated [125I]12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added 125I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of [125I]12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus

Juvenile hormone binding proteins (JHBPs) were extracted from the haemolymph of adult desert locusts, Schistocerca gregaria, and Mediterranean field crickets, Gryllus bimaculatus. The JHBPs were purified by polyethyleneglycol precipitation, filtration through molecular weight cut off filters and chromatography on a HiTrap heparin column. The juvenile hormone (JH) binding activity of the extracts was measured using a hydroxyapatite assay and the purification progress was monitored by native gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph JHBPs of both insects are hexamers composed of seemingly identical subunits. The JHBP of the locust has a native Mr of 480 kDa with subunits of 77 kDa, whereas the JHBP of the cricket has a Mr of 510 kDa with subunits of 81 kDa. The locust JHBP binds JH III with moderate affinity (KD = 19 nM). Competition for binding of JH II and JH I was about 2 and 5 times less, respectively. The cricket JHBP also has a moderate affinity for JH III (KD = 28 nM), but surprisingly, competition for binding of JH II was equal to that of JH III and JH I competed about 3 times higher. No sequence information was obtained for the locust JHBP, but the N-terminal sequence of the cricket JHBP shows ca. 56% sequence homology with a hexamerin from Calliphora vicina. Antisera raised against the purified JHBPs were used to measure age- and sex-dependent changes in haemolymph JHBP titres and to confirm that the JHBPs of both species are immunologically different.     

Synthesis and secretion of a precursor hemolymph juvenile hormone-binding protein in the adult female cockroach Leucophaea maderae

Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae. The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro, a JH-binding component was found that is JH III saturable, has a K_D of 1.5 × 10⁻⁸ M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoaffinity labeled with 10-[10,11-³H]epoxyfarnesyl diazoacetate ([³H]EFDA). The results revealed that two media proteins bound [³H]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000.To determine if the JHBP found in media of fat body cultures was due to hemolymph contamination of fat body, incorporation of [³H]leucine into newly synthesized and secreted fat body proteins during a 48 h culture period was monitored. During the culture period, linear increases in the concentrations of radiolabeled 275 and 220 kD JHBP were observed. Monoclonal antibodies specific for the 220 kD hemolymph JHBP were found to recognize both the 275 and 220 kD JHBPs in the media.To investigate the possibility that the 275 kD protein is a precursor to the 220 kD protein and that components of the hemolymph process or modify the precursor, hemolymph was introduced into fat body cultures and relative concentrations of the 275 and 220 kD media JHBPs were determined. Addition of hemolymph to these organ cultures resulted in an increase in the concentration of radiolabeled 220 kD JHBP and a proportional decrease in the concentration of radiolabeled 275 kD JHBP, suggesting that the 275 kD protein is a precursor to the 220 kD hemolymph JHBP. The mechanism of processing or modification remains undetermined.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

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 K_d 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.     

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

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r 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 M_r 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

Juvenile 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.     

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.     

Cytosolic and nuclear receptors for juvenile hormone in fat bodies of Leucophaea maderae

Cytosol preparations of fat bodies from adult Leucophaea maderae contained a population of very high affinity JH binding compounds (K_d of 10⁻⁹ M) which was only identifiable by the dextrancoated charcoal assay. These compounds exhibited a 1.5 times higher affinity to the natural enantiomer (10R-JH III) than to the racemate. A binding compound for JH III with similar affinity and identical sedimentation characteristics on sucrose gradients could be extracted from isolated nuclei of only vitellogenic fat bodies, either natural or (RS)-methoprene induced. This high affinity JH binder could not be extracted from nuclei of fat bodies from males except those males which had been treated with the JH analogue. These same males were induced to synthesize vitellogenin. A population of lower affinity JH binders (K_d of 10⁻⁸ M) was identified in cytosol and nuclear extracts by the DCC assay procedure as well as by the polyethylene glycol and hydroxylapatite assays. We conclude that the high affinity JH binder of cytosol and nuclei of fat bodies is the JH receptor of this species.     

Juvenile hormone esterases of Lepidoptera II. Isoelectric points and binding affinities of hemolymph juvenile hormone esterase and binding protein activities

Summary The juvenile hormone esterase (JHE) and juvenile hormone binding protein (JHBP) activities from the last larval instar of 14 species of Lepidoptera (Pieris rapae, Colias eurytheme, Danaus plexippus, Junonia coenia, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Trichoplusia ni, Heliothis virescens, Orygia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea) were analyzed by analytical isoelectric focusing (IEF). While the multiplicity and isoelectric point of these proteins varied, all of them were mildly acidic (pI 4.0–7.0), and a large number of the species possessed only a single JHE and/or JHBP activity. The Michaelis constants (K _m's) of the whole hemolymph JHE activities from selected species for JH III were in the range of 10^–7M. The equilibrium dissociation constantK _d of the JHBP was determined by Scatchard analysis for selected species as well, with the majority of species having aK _d near 10^–7M. This information is consistent with JHE acting as a scavenger for JH at various times during development and relying entirely on mass action to remove JH from its protective JHBP complexes. The JHBP should limit nonspecific binding and thus facilitate the rapid transport of the intact hormone through-out the hemocoel. These data indicate that the species currently used in the study of the developmental biology of the Lepidoptera are biochemically similar to a variety of other species in this order.Abbreviations JH juvenile hormone - JHE juvenile hormone esterase - JHBP juvenile hormone binding protein - IEF isoelectric focusing - EPPAT O-ethyl-S-phenyl phosphoramidothiolate - DFP O O-diisopropyl phosphofluoridate     

Unfolding and refolding of juvenile hormone binding protein

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).     

Identification of specific interaction of juvenile hormone binding protein with isocitrate dehydrogenase

Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.     

Cellular retinol-binding protein from rat liver. Purification and characterization

Cellular retinol-binding protein has been purified to homogeneity from rat liver. The procedures utilized in the purification included acid precipitation, gel filtration on Sephadex G-75 and G-50, and chromatography on DEAE-cellulose. The binding protein was purified approximately 3,500-fold, based on total soluble liver protein. The protein is a single polypeptide chain with a molecular weight of 14,600 based on information obtained by the techniques of sedimentation equilibrium analysis, gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinol with high affinity; the appparent dissociation constant was determined by fluorometric titration to be 1.6 X 10(-8) M. Retinol bound to the protein has an absorption spectrum (lambdamax, 350 nm) considerably altered from the spectrum of retinol in ethanol (lambdamax, 325 nm).     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity.     

Purification and physico-chemical properties of the sex steroid-binding globulin of human blood

A new technique for purification of the human sex hormone-binding globulin (SHBG) is described. This technique includes affinity chromatography of blood serum on cortisol-Sepharose, (NH4)2SO4 fractionation, gel filtration on a Bio-Gel P-300 column and chromatography on a concanavalin A-Sepharose 4B column. From 21 of retroplacental serum 10 mg of pure SHBG (25% yield) has been obtained. Upon gel filtration SHBG behaved as a biopolymer with Mr of 120,000. The molecular weight of SHBG as determined by electrophoresis was shown to be equal to 50,000. SHBG has a sedimentation constant of s20, w of 4.7S, pI of 5.75, extinction coefficient A1%(280,1cm) = 10,5 and association constants of 4.5 X 10(8) and 3.5 X 10(6) M-1 for 5 alpha-dihydrotestosterone and cortisol, respectively. The amino acid and carbohydrate contents of SHBG were determined.     

Characterization and comparison of membrane-associated and cytosolic cAMP-dependent protein kinases. Studies on human erythrocyte protein kinases.

Cyclic AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified, and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit molecular weight of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. These data suggest an asymmetric tetrameric (R2C2) structure (Mr approximately equal to 160,000) for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit molecular weight, pI values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 microgram/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but are clearly dissimilar from those of the soluble type II enzyme. Moreover, regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit molecular weight, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the present results suggest that the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.