Uptake of high-density lipoprotein by Y-organs of the crab cancer antennarius. II. formal characterization of receptor-mediation with isolated membranesкод для вставкиСкачать
Archives of Insect Biochemistry and Physiology 30:77-91 (1 995) Uptake of High-Density Lipoprotein by Y-Organs of the Crab, Cancer antennarius. II. Formal Characterization of ReceptorMediation With Isolated Membranes B y o u n g K. K a n g a n d Eugene Spaziani Depavtment of Biological Sciences, Univessity of lozua, lowa City, Iowa Y-organs are the ecdysial glands of crustaceans, responsible for synthesis and secretion of ecdysteroid hormones. For this purpose, the glands acquire cholesterol as obligate precursor entirely from circulating high-density lipoprotein (HDL). A preceding study provided evidence for the mechanism of acquisition: Y-organs take up cholesterol bound to HDL by an energy-requiring process, receptor-mediated absorptive endocytosis. The present study characterized the receptors involved utilizing isolated Y-organ membranes. HDL binding was saturable and specific; a dissociation constant (KJ of 1.08 x lo-’ M and a binding maximum at equilibrium (Bmax)of 70 pg tHDL protein/mg membrane protein, were obtained. Binding was decreased by protease and was dependent upon calcium. Y-organs are regulated negatively by a peptide hormone from the eystalks, molt-inhibiting hormone (MIH). Y-organ membranes from de-eyestalked crabs (MIH absent) exhibited the same Kd value as membranes from intact crabs, but a B ,, 17% higher. Thus, MIH activity apparently does not change the binding affinity of HDL, but decreases the number of binding sites. These results agree with our previous findings that M I H depresses ecdysteroid synthes i s in part by inhibiting cholesterol uptake. Generally, Y-organ cells appear to contain receptors for HDL that are of high affinity and high binding capacity, similar to the characteristics reported for the binding of insect HDL (vitellogenin) to fat bodies and oocytes. o 1995 WiIey-I.iss, Inc. Key words: Cancer antennarius, cholesterol, ecdysteroids, high-density lipoproteins, receptors, Y-organs INTRODUCTION In crustaceans, a pair of epithelioid glands (Y-organs) secrete steroid hormones (ecdysteroids) that control molting/growth cycles and other vital functions (Skinner, 1985).Y-organ activity is governed by a neurosecretory peptide Received September 15, 1994; accepted March 26, 1995. Address reprint requests to Eugene Spaziani, Ph.D., Department of Biological Sciences, University of lowa, lowa City, IA 52242. 0 1995 Wiley-Liss, Inc. 78 Kang and Spaziani (molt-inhibiting hormone, MIH*) released from ganglionic extensions of the brain in the eyestalks. Control by MIH appears to be novel among known steroidogenic systems (including those of insects and vertebrates) in that MIH inhibits glandular function (Spaziani et al., 1989, 1994; Watson et al., 1989). This two-hormone axis is under investigation as a simpler model for study, at cellular and molecular levels, of cholesterol transport, steroid hormone biosynthesis, and neurohormonal regulation of these processes. Y-organs take up cholesterol as the obligate precursor for ecdysteroid synthesis (Teshima, 1971; Watson and Spaziani, 1985b), and the glands of intact crabs entering premolt take u p greater amounts of ['4Cl-cholesterol than in other stages (Spaziani and Kater, 1973). The same effect is rapidly induced by removing the eyestalks (MIH) (Spaziani et al., 1982; Vensel et al., 1984). Addition of eyestalk extracts to Y-organs in vitro suppresses not only cholesterol uptake from the medium (Watson and Spaziani, 1985b) but also incorporation into an early intermediate (7-dehydrocholesterol) (Rudolph and Spaziani, 1992; Spaziani and Wang, 1993) and ecdysteroid secretion (Mattson and Spaziani, 1985; Soumoff and O'Connor, 1982; Watson and Spaziani, 1985a,b). Thus, several lines of evidence indicate that MIH suppresses Y-organ metabolism and function during intermolt and postmolt but not during premolt, and MIH activity is involved in more than one cellular site or molecular process. It has been determined that Y-organs take up cholesterol against an apparent concentration gradient of sterol (Spaziani and Kater, 1973; Spaziani and Wang, 1993; Vensel et al., 1984). Since Y-organs obtain cholesterol only from circulating high-density lipoproteins (HDL) (Spaziani et al., 19861, the likelihood exists of regulation also by receptors for apoproteins of the cholesterol carrier. In a companion article (Kang and Spaziani, 1995) evidence was presented for (1) the presence of HDL receptors on Y-organ cells, (2) the uptake of the entire HDL-cholesterol complex by adsorptive endocytosis, and (3) inhibition by MIH activity of uptake as well as processing of the complex. Those studies are extended here to a detailed characterization of the presumptive HDL receptors using isolated Y-organ membranes. The findings include the demonstration of high-affinity binding sites for HDL and the suggestion that these are diminished in number by MIH activity. MATERIALS AND METHODS Animal Maintenance Female rock crabs, Cancer antennarius, were maintained in a marine invertebrate facility, temperature controlled (at 10°C) with light-dark cycles (a 12 h light/l2 h dark photoperiod) and in large, self-contained, fiberglass water tables. Sea water was aerated by vertical pumping and filtered through alternating layers of charcoal and crushed oyster shell. In this system, crabs fed every other day with fish were maintained in good health. The organ donors were intact controls and de-eyestalked crabs. De-eyestalking removed the *Abbreviations used: HDL = high-density lipoprotein; LDL = low-density lipoprotein; M I H = molt-inhibiting hormone. High-Density Lipoprotein and Y-Organ Membranes 79 sources of MIH. After chilling crabs on ice, the eyes were excised at the base and the sockets were cauterized. Y-organs were removed and used 48 h later, by which time the Y-organs are activated in induced premolt. Preparation of lZ5I-HDL The total HDL fraction was prepared from crab hemolymph (Watson and Spaziani, 198513). The concentrated HDL was labeled with 1251by the iodomonochloride method of McFarlane (1958) as modified by Langer et al. (1972). The essential details of these procedures are presented in this issue’s companion paper (Kang and Spaziani, 1995). The iodinated HDL had a specific activity that averaged 8 x lo6DPM/mg of protein. Chemicals Protease was purchased from Sigma Chemical Co. (St. Louis, MO). Sucrose was from Fisher Scientific Company (Fair Lawn, NJ). 2-Amino-2-(hydroxymethyl)-1,3-propandiol-hydrochloride,(Tris-HC1) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN), 125I was from Amersham Corp. (Arlington Heights, IL). Preparation of Y-Organ Membranes The procedure described is based on that of Rudolph and Spaziani (1992), which accommodates crustacean organelles, but is modified to maximize purity of the plasma membrane fraction. Y-organs from intact or de-eyestalked crabs were held in Pantin’s saline (Pantin, 1934) at 4°C. All further operations were carried out at 4°C. Y-organs were homogenized in a 7 ml Dounce Tissue Grinder (Wheaton Scientific, Millville, NJ) in homogenization buffer (0.528 M sucrose, 2.5 mM CaC12,and 10 mM Tris-HC1, pH 7.4). The homogenate was centrifuged at 1OOg for 10 min. The resulting supernatant was centrifuged at 1,OOOg for 10 min. The 1,OOOg pellet was resuspended in 2.2 M sucrose by flushing 10 times through a micropipette tip and the suspension was then centrifuged at 40,OOOg for 1 h. The 40,OOOg supernatant was resuspended 4 times with resuspension buffer (50 mM NaC1,l mM CaC12,and 20 mM Tris-HC1, pH 7.5), and the suspension was sedimented at 10,OOOg for 10 min. The sedimented pellets were resuspended in resuspension buffer. The pellets were centrifuged at 10,OOOg for 10 min. The final pellets were resuspended in resuspension buffer. The suspensions were then sonicated for 20 s at setting 6 in a Sonifier Cell Disruptor (Model W 185, Heat-Systems Ultrasonics, Inc., Plainview, NY). The homogeneity of prepared Y-organ membranes was monitored by negative staining and electron microscopy. The concentration of Y-organ membrane proteins was measured using BCA protein assay reagent (Pierce, Rockford, IL). Y-organ membranes were stored frozen when necessary. Binding Assay The binding of lz5I-HDLto Y-organ membranes was measured by a modified micro-binding assay based on the macro-methods as previously described for membrane preparation from rat liver (Kovanen et al, 1979b) and from mouse and rat adrenal glands (Kovanen et al., 1980). The standard binding 80 Kang and Spaziani assay was conducted at a final pH of 7.5 in 40 p1 of reaction buffer containing 100 mM NaC1, 0.5 mM CaC12, 50 mM Tris-HC1, 20 mg/ml bovine serum albumin, various amounts of membrane proteins, and with or without indicated amounts of unlabeled HDL. The binding reactions in microcentrifuge tubes were initiated by addition of "jI-HDL and then incubated on ice for the designated length of incubation time. Reactions were terminated by addition of 75 pl of ice-cold filtered fetal calf serum (FCS). The microcentrifuge tubes were centrifuged at 10,OOOg for 5 min at 4°C. The 10,OOOg pellet was rinsed by 100 1-11 of ice-cold filtered FCS. The tubes were recentrifuged at 10,OOOg for 5 min at 4°C and the supernatant was removed. The tips of microcentrifuge tubes containing the pellets were separated from the remainder of the tube by cutting off with a razor blade, and tube tips were counted in a Beckman Model 4000 Gamma Counter (Beckman Instruments, Inc., Palo Alto, CA). Saturability studies were performed by incubating Y-organ membranes (2 ,ug protein/40 1-11 of buffer) obtained from intact or de-eyestalked crabs with various concentrations of 12'I-HDL for 1 h on ice in the absence or presence of 40 pg unlabeled HDL/40 pl of buffer. Specific binding was obtained as the difference of total binding of HDL in the absence of the excess unlabeled HDL and the total binding in the presence of unlabeled HDL. Scatchard plots were constructed by plotting the ratio of specific bound 12'I-HDL to free 12'1HDL vs. specific bound '*'I-HDL (Scatchard, 1949). The time course study of binding of '"I-HDL to Y-organ membranes was performed by incubating 2 pg of membranes obtained from de-eyestalked crabs with 5 pg of '"I-HDL for the indicated length of incubation time on ice in the absence or presence of 40 pg/40 pl of unlabeled HDL. To study the competition for binding of '251-HDLvs. increasing concentration of unlabeled HDL, 5 pg of Y-organ membrane proteins from de-eyestalked crabs were incubated with 5 pg of "'I-HDL for 1 h on ice in the absence or presence of increasing amounts of unlabeled HDL. The relationship between binding of 12?-HDL and increasing concentrations of membrane proteins was examined. Increasing amounts of Y-organ membranes from de-eyestalked crabs were incubated with 5 pg of 12?I-HDL for 1 h on ice in the absence or presence of 40 pg of unlabeled HDL. To illustrate effects of protease on binding of lZ5I-HDL,2 pg of membrane proteins from de-eyestalked crabs were treated with different concentrations of protease, and then incubated for 15 min at 37°C. After incubation, the membrane mixture containing protease was transferred to ice cold water bath and allowed to sit for 5 min to stop the action of protease. Protease-treated membranes were incubated with 5 pg of '251-HDLfor 2 h on ice in the absence or presence of 40 pg of unlabeled HDL. To study effects of ethylenediamine tetraacetic acid (EDTA) and calcium on binding of '251-HDL,Y-organ membranes from de-eyestalked crabs were prepared as described previously except that calcium was omitted from homogenization, resuspension, and reaction buffers. Y-organ membranes (2 pg) were incubated with different concentrations of EDTA for 2 h on ice in the absence or presence of 40 pg of unlabeled HDL. For the determination of effects of calcium on binding of 12'I-HDL, Y-organ membranes (2 pg) were High-Density Lipoprotein and Y-Organ Membranes 81 incubated with 1 mM EDTA, indicated amounts of calcium, and 5 pg/40 p1 of '251-HDLfor 2 h on ice in the absence or presence of 40 pg of unlabeled HDL. RESULTS Saturability of Binding and Scatchard Analyses The binding of '''I-HDL to Y-organ membranes in the absence or presence of excess unlabeled HDL was measured (Fig.1). The difference between the 0 1000 2 4 2 4 6 8 10 12 - 0 6 8 1 0 1 25 I-HDL (x 10" M) 2 Fig. 1. Saturation curves for the binding of '251-HDLto Y-organ membranes. Purified Y-organ membranes (2 pg protein) from de-eyestalked (A) and intact (B) crabs were incubated in 40 pI with indicated amounts of '251-HDL in the absence or presence of 40 pg of unlabeled HDL. After incubation for 1 h at 4"C, the amount of 12'1-HDL bound to membranes was determined. The specific binding (filled squares) was obtained by subtracting the amount of non-specific binding (open circles) from that of total binding (open squares). All values are means (n = 6). 82 Kang and Spaziani total "jI-HDL binding in the absence of unlabeled HDL and non-specific 1251HDL binding in the presence of unlabeled HDL gave a measure of the specific binding. The plot of the specific binding demonstrated saturability at 5 pg/40 yl of '"1-HDL. It was apparent that specific binding of '251-HDLto Yorgan membranes from de-eyestalked crabs was saturable, whereas non-specific binding increased slightly in linear fashion with increasing "jI-HDL ligand concentration, indicating that lZ5I-HDLbinding to membranes was inhibited com etitively by the presence of excess unlabeled HDL. The concentration of I2$-HDL giving half-maximal binding (Kd) to Y-organ membranes from de-eyestalked crabs was about 20 pg/ml of '251-HDLprotein. This is a Kd of 1.08 x M calculated from a Scatchard plot (Fig. 2) of these data and based on a molecular weight of 185,000 for crab HDL apoprotein (Spaziani et al., 1986). The maximal binding (Bmax)at equilibrium was about 0.07 yg of lZ51-HDLprotein/pg of membrane protein (70 yg/mg). The Kd of '251-HDLto Y-organ membranes from intact crabs (Fig. 1B) was about 20 yg/ml of 1251HDL and the B,, was 0.06 yg of 12jI-HDLprotein/pg of membrane protein (60 yg/mg). These data indicate that Y-organ membranes from intact and deeyestalked crabs have the same binding affinity of '=I-HDL but that membranes from de-eyestalked crabs possess about 1.2-fold more HDL receptor sites than Y-organ membranes from intact crabs. The similarity of binding affinity of lZ51-HDLto membranes from both groups and the indication of the presence of more HDL receptors in Y-organ membranes from de-eyestalked crabs were apparent when the specific binding data of two groups were plotted according to the methods of Scatchard (1949) (Fig. 2). The two Scatchard plots showed parallel slopes, supporting the conclusion that Y-organ membranes from the two groups have the same binding affinity to '251-HDL.The ratio of high affinity sites of membranes from de-eyestalked crabs to sites of membranes from intact crabs was about 1.2. The Scatchard analysis suggests that the suppressed HDL uptake and degradation by Y-organs from intact 0.14 0.10 0.08 0.06 0.04 0.02 0.00 Fig. 2. Scatchard plots of specific binding data of Y-organ membranes from de-eyestalked (open circles) and intact crabs (filled circles). (There are seven open circles, of which two fall on the line and appear to be filled.) High-Density Lipoprotein and Y-Organ Membranes 0 60 120 83 180 Incubation time (min) Fig. 3. Time course of binding of 12'1-HDL to Y-organ membranes from de-eyestalked crabs. Membrane protein (2 pg) was incubated in 40 ~l with 5 pg of '*'I-HDL in the absence (open circles) or presence (filled circles) of 40 pg of unlabeled HDL. After incubation at 4°C for the indicated length of incubation, the amount of membrane-bound '251-HDL was measured. All values are means (n = 10). Means between the two curves were significantly different from 5 to 180 min (P < 0.001). crabs (MIH-present) observed previously was due at least in part to regulation of numbers of HDL receptor sites by MIH. Time Course of Binding of lz5I-HDLto Y-Organ Membranes The time course of binding was studied with Y-organ membranes from deeyestalked crabs (Fig. 3). At the first time point (5 min), non-specific binding was essentially maximal. When membranes were incubated for 180 min in the presence of 40 pg of unlabeled HDL, the total binding of Iz5I-HDLto membranes was reduced by 80%. The results confirm the presence of specific saturable binding sites for HDL. Effects of Increasing Concentrations of Unlabeled HDL on Binding of lZ5I-HDL The competition by unlabeled HDL with "'I-HDL for membrane binding was examined by the incubation of Y-organ membranes from de-eyestalked crabs for 1 h at 4°C with 5 pg of lz5T-HDLin the presence of increasing concentration of unlabeled HDL (Fig. 4). The binding of "'I-HDL was inhibited competitively as concentration of unlabeled HDL increased. At an unlabeled HDL concentration of just 4 pg, 50% inhibition was achieved. At concentrations over 40 pg of unlabeled HDL, inhibition did not further change (data not shown). Effects of Increasing Concentrations of Y-Organ Membranes on Binding of lZ5I-HDL Binding was measured using 1 to 5 pg/40 pl of Y-organ membrane protein from de-eyestalked crabs (Fig. 5). The amount of l2'1-HDL binding rose lin- 84 Kang and Spaziani 10 20 30 unlabeled HDL (Ug/40 ul) 0 40 Fig. 4. Binding of ''?I-HDL to Y-organ membranes in the presence of increasing concentrations of unlabeled HDL. Each incubation tube (40 yl) contained 5 pg of membrane protein from deeyestalked crabs, 5 pg of '"I-HDL, and indicated amounts of unlabeled HDL. After incubation at 4°C for 1 h, the amount of '"I-HDL bound to the membranes was measured. Points represent the percentage of binding relative to control taken as 100%. Controls contained no unlabeled HDL (n = 10). early in proportion with increasing amounts of membrane protein in the binding assay as predicted. The binding of '"I-HDL to Y-organ membranes was inhibited competitively by excess unlabeled HDL. The binding of 12'I-HDL to membranes in the presence of unlabeled HDL was reduced by approximately 80% at all membrane concentrations used. The results are consistent with the 75m ---- . 2000 - 1500 - 1000 500 0.0 1.0 2.0 3.0 4.0 Membrane protein (ug/40 ul) 5.0 Fig. 5. Binding of '151-HDL to Y-organ membranes as a function of increasing concentrations of '251-HDL and indicated of Y-organ membrane protein. Each incubation (40 pl) contained 5 1-18 amounts of membrane protein from de-eyestalked crabs in the absence (open circles) or presence (filled circles) of 40 yg of unlabeled HDL. After incubation at 4°C for 1 h, the amount of "'1-HDL bound to the membranes was measured. Points are means (n = 1 0 ) . High-Density Lipoprotein and Y-Organ Membranes 85 presence of HDL receptors in Y-organ membranes as indicated by the straight line relationship. Effects of Protease on Binding of "'1-HDL to Y-Organ Membranes To find if modification of Y-organ membrane surface proteins affects 125IHDL binding to membranes, Y-organ membranes from de-eyestalked crabs were treated with different concentrations of protease. After treatment with protease for 15 min, membranes were incubated with 5 pg of I2'I-HDL in the absence or presence of 40 pg of unlabeled HDL. Protease treatment decreased binding significantly at all concentrations of the enzyme used (Fig. 6). Maximum inhibition (72%) was 0.6 pg of protease, without further change with 1 pg. Non-specific binding was not affected. These data are consistent with the presence of specific proteinous binding sites (receptor) for HDL on Y-organ membranes. Effects of EDTA and Calcium on Binding of lZ5I-HDLto Y-Organ Membranes The binding of '"I-HDL to Y-organ membranes was affected by the calcium concentration in the incubation medium (Fig. 7). When membranes from de-eyestalked crabs were incubated with the calcium chelator EDTA, the high affinity binding of '251-HDLwas inhibited without affecting the non-specific binding. At incubation of membranes with 0.2 mM of EDTA, the binding of '251-HDLto Y-organ membranes was maximally inhibited approximately 50% without further significant inhibition to 1.0 mM (Fig. 7A). The addition of calcium overcame the EDTA inhibition and restored the high-affinity binding of '*'I-HDL to Y-organ membranes without affecting non-specific binding - .-Ca 2000 1500 ti n 5E 1000 M a 2 e 500 v o ! . 1 0.0 0.2 . 1 0.4 - 1 0.6 . 1 0.8 . I 1.0 Protease (ug/40 ul) Fig. 6. Effects of protease on '"I-HDL binding to Y-organ membranes. Y-organ membranes from de-eyestalked crabs were incubated for 15 min at 37°C .with indicated concentrations of protease. The action of protease was stopped by immediate chilling of incubation tubes in icecold water bath for 5 min. After protease treatment, the binding assay was performed. Each tube contained 40 pI of buffer, 2 pg of membranes, and 5 pg of '"I-HDL in the absence (open circles) or presence (filled circles) of 40 pg of unlabeled HDL. After incubation for 2 h at 4"C, the amount of '251-HDL bound to the membranes was determined. Points are means (n = 6 ) . 86 Kang and Spaziani -2 500 - 1 2000 " -- -- a m - a 0 v (B) I 0 . I 5 - I 10 - I 15 - I 20 - I 25 Concentration of Calcium (mM) Fig. 7. Effects of EDTA and calcium on binding of '251-HDL to Y-organ membranes. Y-organ membranes from de-eyestalked crabs were prepared as described under Materials and Methods, except that calcium was omitted from all solutions required for the preparation of the membranes. Each incubation tube contained 2 pg of membranes, 5 pg of '*'I-HDL, and indicated amounts of EDTA in the absence (open circles) or presence (filled circles) of 40 pg of unlabeled HDL. After incubation for 2 h at 4"C, the amount of '251-HDLbound to the membranes was determined (A). In an experiment to show the effect of calcium on binding of '*'I-HDL to Yorgan membranes (B), each incubation tube contained 2 pg of membranes prepared as described above, 1 m M EDTA, 5 pg of I2'1-HDL, and indicated concentrations of calcium i n the absence (open squares) or presence (filled squares) of 40 pg of unlabeled HDL. After incubation for 2 h at 4"C, the amount of '"I-HDL bound to the membranes was determined. Points are means (n = 7). High-Density Lipoprotein and Y-Organ Membranes 87 (Fig. 7B). In the presence of EDTA and 0 calcium (point on ordinate of Fig. 7B), binding of HDL was not significantly different than non-specific binding. With addition of 0.5 mM calcium, the binding of 12'1-HDL was rapidly increased to the maximal level. This study demonstrates that calcium is required for the binding of HDL to Y-organ membranes. DISCUSSION The present study characterizes the presence of specific binding sites for HDL in Y-organ plasma membranes. The Y-organ membranes from both intact and de-eyestalked crabs exhibited saturability of HDL binding with the M. The maximal HDL binding same dissociation constant (Kd) of 1.08 x activity of membranes (BmaX:70 pg/mg) from de-eyestalked crabs (MIH absent) was 17%higher than observed by Y-organ membranes from intact crabs (BmaX:60 pg/mg) (Figs. 1 and 2). These results suggest that MIH decreases HDL binding activity of membranes by decreasing the number of HDL binding sites, not the receptor affinity. In addition, these data indicate the presence generally of HDL receptors with relatively high binding affinity. The lower number of sites of membranes obtained from intact crabs agrees with previous results that eyestalk MIH inhibited total HDL uptake (Kang and Spaziani, 1995) and uptake of cholesterol (Spaziani and Wang, 1993; Watson and Spaziani, 19854. The decrease in HDL receptor sites could be related to the MIH-induced inhibition of protein synthesis in Y-organs as shown in a previous study (Mattson and Spaziani, 1986),which demonstrated that MIH activity suppressed Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level. To our knowledge, analyses of the kind reported here on receptor binding of lipoprotein by the steroidogenic Y-organs have not been done in the analogous structures of insects, the prothoracic glands. However, several studies have characterized vitellogenin (HDL) binding by oocytes. In crustaceans, Laverdure and Soyez (1988) measured binding of vitellogenin HDL to lobster oocyte membrane proteins. An apparent Kd of 7 x lo-' M was obtained and the Kd was not affected by calcium. Interestingly, the number of binding sites was highest at the beginning of vitellogenesis, and then decreased until reduced by 80% in the older oocytes. In insects, HDL binding has been studied in fat bodies and oocytes (for background see Oliveira et al., 1986). Tsuchida and Wells (1990) isolated receptor protein from fat body cell membranes and found a dissociation constant (Kd, binding affinity) of 4.1 x lo-' M (25 pg/ml) for binding of insect hemolymph HDL. Oocytes of Rhodnius showed a Kd of 5 x M and a binding capacity (BmaX)of 600-900 pg of HDL protein/mg of membrane protein (Wang and Davey, 1992); a similar Kd value (1.1 x M) was obtained for locust (Locusta) oocyte membranes, but the B,, was 20 pg/mg (Rohrkasten and Ferenz, 1986). Thus, for arthropod systems so far studied, receptor systems that bind HDL appear to be quite similar and can be categorized generally as high affinity, high capacity. In contrast, vertebrate lipoprotein receptors (in steroidogenic cell membranes) can be described as high affinity, low capacity. For example, mouse adrenal cortical membranes exhibited a Kd of 8-10 pg/ml and a B,, of only 0.29 pg LDL 88 Kang and Spaziani protein/mg membrane protein (which rose to 0.62 after ACTH treatment) (Kovanen et al., 1980); similarly, the values for bovine adrenals were Kd 15 yg/ml and 1-2 yg/mg for homologous LDL (Kovanen et al., 1979a). For LDL receptor of corpora lutea, binding affinities are similar in human (Ohashi et al., 1982) and rat (Hwang and Menon, 1983) at Kd of 8-11 pg/ml with binding capacities of 0.062 and 2.8 pg/mg, respectively. Interestingly, receptors for HDL in the luteal cells showed somewhat higher Kd (lower affinity) than L,DL., 136 and 18 ,ug/ml, respectively, reminiscent of the values for HDL binding in arthropods. I n other respects, the mechanism of HDL-cholesterol uptake by Y-organ cells resembles that of classical LDL-cholesterol uptake by mammalian steroidogenic cells of the adrenal cortex (Brown et al., 1979; Kovanen et al., 19801, ovary (Ohashi et al., 1982; Rajan and Menon, 1987; Rajkumar et al., 1989), and testis (Benahmed et a]., 1983; Freeman and Ascoli, 1982). That is, cholesterol as precursor is preferentially acquired by endocytosis of the lipoprotein carrier, a process that is receptor mediated and sensitive to proteolysis, calcium, and regulatory tropins. The tropins regulate by increasing the number of receptor sites. The mammalian receptors involved feature specific high-affinity binding for apoprotein B of LDL. The same receptors also bind apoprotein E-containing HDL, but usually with less affinity and where cholesterol is delivered significantly, the mechanism appears to be different than for LDL (Gwynne and Strauss, 1982; Rajkumar et al., 1989). Exceptions occur in rodents in which (1) HDL, not LDL, is the predominant circulating lipoprotein, (2) HDL is the primary source of cholesterol (Gwynne and Strauss, 1982), (3) specific receptors occur for the A-I and A-I1 varieties of apoproteins on HDL (Hwang and Menon, 1985), which are not sensitive to proteolysis or calcium (Hwang and Menon, 1983), and (4) liberation of cholesterol differs in that it is not necessarily dependent upon breakdown of the complex by lysosomes (Gwynne and Mahaffee, 1989; Rajkuniar et al., 1989; Schreiber et al., 1982) nor upon endocytosis (Nestler et al., 1985; Pittman et al., 1987; Rajan and Menon, 1989; Schreiber et al., 1982). Thus, in comparing lipoprotein uptake mechanisms for steroidogenic glands between crustaceans and mammals, the Y-organs d o not fully conform to either the LDL or the rodent HDL models. Rather, the crustacean system presents a hybrid mechanism. Resemblance with the LDL path includes the presence of specific receptors, moderately high-affinity binding, sensitivity of binding to proteolysis, calcium and tropin regulation, endocytosis (Kang and Spaziani, 1995), and an HDL apoprotein that shares some physicochemical characteristics with apoprotein B of LDL (Spaziani et al., 1986). Resemblance with the rodent HDL model includes the dominance of HDL in the circulation (actually, the only lipoprotein), HDL as the source of cholesterol, and intracellular HDL degradation without apparent interaction with lysosomal enzymes (Kang and Spaziani, unpublished data). Unusual, if not unique features in Y-organs are an absence of cholesterol storage or d e novo synthesis, HDL receptors of high binding capacity and negative regulation of receptor sites by the peptide tropic hormone. 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