Intramolecular group transfer is a characteristic of neurotoxic esterase and is independent of the tissue source of the enzyme. A comparison of the aging behaviour of di-isopropyl phosphorofluoridate-labelled proteins in brain, spinal cord, liver, kidney and spleen from hen and in human placenta

Neurotoxic esterase activity was measured in homogenates of human placenta and hen brain, spinal cord, liver, kidney and spleen. The activity in liver comprised less than 20% of the Paraoxon-resistant esterases, but in the other tissues neurotoxic esterase accounted for over 50%. The same tissues were labelled with [3H]di-isopropyl phosphorofluoridate, and any isopropyl group transferred on to protein during 'aging' of the labelled enzymes (alkali-volatilizable tritium) was measured. No Paraoxon-sensitive labelled sites were found to age in this way in any tissue. In brain, the Paraoxon-resistant alkali-volatilizable-tritium-labelled sites correlated with the number of neurotoxic esterase labelled sites, indicating that 'aging' and isopropyl group transfer were 100% efficient. The site receiving the transferred isopropyl group was characterized by analysing the distribution of radiolabelled proteins on gel-filtration chromatography in the presence of SDS. In particulate preparations from each tissue, the protein-bound alkali-volatilizable tritium (transferred isopropyl group) was attached to a polypeptide of Mr 178 000. This same polypeptide also bore the isopropyl-phosphoryl group of neurotoxic esterase, indicating that aging of neurotoxic esterase is an intramolecular group transfer. The apparent turnover number for the enzyme (average 1.6 X 10(5) min-1) was approximately the same in each hen tissue, confirming that closely similar enzymes were present in brain, spinal cord, liver and spleen. The apparent turnover for the human enzyme was 1.8-fold higher than that for the hen enzyme. The concentration of the neurotoxic esterase phosphorylated subunit in brain, spinal cord, spleen, placenta and liver was 14.6, 3.8, 7.4, 3.3 and 3.8 pmol/g of tissue. The evidence indicated that neurotoxic esterase is present in each tissue except kidney, and that isopropyl group transfer on 'aging' occurs on this enzyme only. This process is an intramolecular transfer of the group within the same polypeptide.     

A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds

1. It is proposed that part of a neurotoxic dose of di-isopropyl phosphorofluoridate will be covalently bound in vivo to a specific component in the brain and spinal cord as the initial biochemical event in the genesis of the lesion. 2. A test system in vitro was devised that removes many di-isopropyl phosphorofluoridate-binding sites and indicates that the specific component may be a protein present in brain at a concentration comparable with that of the cholinesterases. 3. The site was found to be present and capable of binding di-isopropyl phosphorofluoridate in vitro in brain samples taken from either normal hens or those dosed with organophosphorus esterase inhibitors that are not neurotoxic. 4. Very little of the specific binding activity was found in brain samples from hens pre-dosed with a variety of neurotoxic organophosphorus compounds. 5. A solubilized preparation of the active brain component was obtained, suitable for further purification and study.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Solubilization of cerebral binding sites of S-adenosyl-L-homocysteine

We describe the solubilization of S-adenosyl-L-homocysteine binding sites from rat brain membranes; Triton X100 could solubilize near 50% of the sites. The solubilized extract exhibited the same pH dependence as the membrane extract and had the same dissociation constant and the same sensibility to S-adenosyl-L-methionine and adenosine. The solubilized extract exhibited a methylase activity which accepted phosphatidylethanolamine as substrate.     

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.     

Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.     

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.     

The metabolism of neuropeptides. Phase separation of synaptic membrane preparations with Triton X-114 reveals the presence of aminopeptidase N.

The property of solutions of Triton X-114 to separate into detergent-rich and detergent-poor phases at 30 degrees C has been exploited to investigate the identities of the aminopeptidases in synaptic membrane preparations from pig striatum. When titrated with an antiserum to aminopeptidase N (EC 3.4.11.2), synaptic membranes solubilized with Triton X-100 revealed that this enzyme apparently comprises no more than 5% of the activity releasing tyrosine from [Leu]enkephalin. When assayed in the presence of puromycin, this proportion increased to 20%. Three integral membrane proteins were fractionated by phase separation in Triton X-114. Aminopeptidase activity, endopeptidase-24.11 and peptidyl dipeptidase A partitioned predominantly into the detergent-rich phase when kidney microvillar membranes were so treated. However, only 5.5% of synaptic membrane aminopeptidase activity partitioned into this phase, although the other peptidases behaved predictably. About half of the aminopeptidase activity in the detergent-rich phase could now be titrated with the antiserum, showing that aminopeptidase N is an integral membrane protein of this preparation. Three aminopeptidase inhibitors were investigated for their ability to discriminate between the different activities revealed by these experiments. Although amastatin was the most potent (IC50 = 5 X 10(-7) M) it failed to discriminate between pure kidney aminopeptidase N, the total activity of solubilized synaptic membranes and that in the Triton X-114-rich phase. Bestatin was slightly more potent for total activity (IC50 = 6.3 X 10(-6) M) than for the other two forms (IC50 = 1.6 X 10(-5) M). Puromycin was a weak inhibitor, but was more selective. The activity of solubilized membranes was more sensitive (IC50 = 1.6 X 10(-5) M) than that of the pure enzyme or the Triton X-114-rich phase (IC50 = 4 X 10(-4) M). We suggest that the puromycin-sensitive aminopeptidase activity that predominates in crude synaptic membrane preparations may be a cytosolic contaminant or peripheral membrane protein rather than an integral membrane component. Aminopeptidase N may contribute to the extracellular metabolism of enkephalin and other susceptible neuropeptides in the brain.     

Partial characterization of neuropathy target esterase and related phenyl valerate esterases from bovine adrenal medulla

The mechanism by which organo-phosphorus-induced delayed polyneuropathy is induced relates to the specific inhibition and subsequent modification (“aging”) of a protein known as neuropathy target esterase (NTE), operatively defined as paraoxon-resistant and mipafox-sensi-tive phenyl valerate (PV) esterase activity. This protein has fundamentally been investigated in hen brain, the latter being the habitually employed OPIDP study model. In the present article, a partial characterization is made of the NTE and other related PV esterases in the bovine adrenal medulla and brain; NTE sensitivity to the neurotoxic or-ganophosphorus compound mipafox is investigated, and its subcellular distribution is studied. The NTE activity of the adrenal medulla was found to be the highest of those among the tissues studied to date (5000 ± 1400 mU/g tissue; ± SD, n = 12). This activity represented 93% of the PV esterase activity resistant to 40 μm paraoxon in the par-ticulate fraction of the adrenal medulla and approximately 50% of total PV esterase activity. In the bovine brain, these proportions were 72 and 26%, respectively, i.e., similar to those described in hen brain. The mipafox inhibition curve of PV esterase activity resistant to 40μM paraoxon in the particulate fraction of the adrenal medulla suggests that NTE activity fundamentally comprises a mipafox-sensitive component with an I ₅₀ of 6.39 μM at 30 minutes, which is similar to the value reported in hen brain. NTE activity in the bovine adrenal medulla is almost exclusively limited to the particulate fraction, the microsomal fraction, plasma membrane, and chromaffin granule-enriched fractions being the highest in terms of specific activity. On the contrary, the mitochondria-enriched fraction was very poor in such activity. In bovine brain, most NTE activity was likewise limited to the particulate fraction.     

Cholesterol ester hydrolase(s) in mammalian brain: Is there a myelin-specific cholesterol ester hydrolase?

The present study compared the properties of cholesterol ester hydrolase(s) in myelin and microsomes from rat, mouse and human brain. The results indicated that the enzyme activity in both myelin and microsomes from rat, mouse and human brain was optimal at pH 6.5 and required Triton X-100 for optimal activity. The enzyme activity in myelin was 3- to 4-fold higher in the presence of Trition X-100 than taurocholate. Addition of phosphatidyl serine enhanced (2 to 4 fold) the hydrolase activity in both myelin and microsomes. The properties of the enzyme in solubilized preparation of myelin were also similar to the properties of the enzyme in partially delipidated and solubilized preparations of microsomes. The activity was again optimal at pH 6.5, required Triton X-100 for optimal activity and was stimulated by phosphatidyl serine. These results indicate that the properties of cholesterol ester hydrolase in myelin are similar to those of the microsomal enzyme and that this is true for the fractions from both human and rodent brain. The data thus lead us to believe that the hydrolase activity in mammalian brain myelin and microsomes may reflect the distribution of a single enzyme in the two fractions rather than two distinct enzymes, one being specific to each fraction.     

Allozyme variation of nonspecific esterases in Russian sturgeon (Acipenser güldenstädti Brandt

Electrophoresis in 7% polyacrylamide gel was used to analyze nonspecific esterases from different organs and tissues of Russian sturgeon. The basic enzymatic activity was observed the four zymogram zones. In sixteen cases the patterns of isozyme distribution in these zones allowed to consider them as allele systems (14 polymorphic and 2 monomorphic) corresponding to the four structural genes. The observed genotype frequencies of the nine of these systems (EST-1* of brain and intestine; EST-2* of serum and intestine; EST-3* of spleen and kidney; and EST-4* of spleen, brain, and heart) were largely concordant with Hardy-Weinberg proportions for codominant disomic inheritance. Genetic control of other polymorphic systems is unknown, but, high enzymatic activity of these esterases, sufficient for qualitative electrophretic detection, permits utilization of these polymorphisms for phenotypic monitoring of Russian sturgeon populations.     

Characteristics of thyrotropin releasing hormone (TRH) receptors in rat brain

Characteristics of TRH-receptors were studied in the rat central nervous system (CNS). Ion species, pH and temperature importantly influenced TRH-receptor binding. Subcellular distribution of TRH-receptor binding revealed that synaptic membranes had the greatest percentage of total sites. Scatchard analysis suggested that the rat CNS had two distinct TRH binding sites with apparent dissociation constants (Kd) of 5 X 10(09) M and 13 X 10(-8) M. Receptor activity is sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for the binding of [3H]TRH. Thiol reagents reduced the binding activity of the receptor, suggesting that an intrachain disulfide bond may form an important constituent of the binding site for TRH. The TRH-receptor in the rat brain was successfully solubilized with non-ionic detergent Triton X-100. On gel chromatography with Sepharose 6B column, the solubilized TRH-receptor molecule eluted at the fraction corresponding to an apparent molecular weight of 300,000 daltons, with Stokes' radius of 5.8 nm. The regional distribution of TRH-receptor binding was examined to clarify the site of TRH action. The highest level of binding was in the hypothalamus, cerebral cortex and hippocampus, indicating that TRH affects the CNS function mainly through the limbic system, cerebral cortex and hypothalamus. Moreover, tricyclic anti-depressants and Li+ decreased the binding of [3H]TRH. These findings suggest that endogenous TRH and TRH receptor may play the role of a neurotransmission modulator in the brain to control emotional and mental functions.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) activity for myelin basic protein was found to be present in the myelin fraction of rat brain. The enzyme activity was in a latent form and solubilized by 0.2% Triton X-100 treatment with about 50% increase of activity. The cytosol fraction from bovine brain also had phosphoprotein phosphatase activity for myelin basic protein, which was resolved into at least two peaks of activity on DEAE-cellulose column chromatography. Myelin basic protein was the best substrate for both the solubilized myelin fraction and the cytosol enzymes among the substrate proteins tested. The Km values of the solubilized myelin fraction were 4.2 muM for myelin basic protein, 7.4 muM for arginine-rich histone, 8.0 muM for histone mixture and 14.3 muM for protamine, respectively.     

The mechanism of action of vinblastine. Binding of [acetyl-3H]vinblastine to embryonic chick brain tubulin and tubulin from sea urchin sperm tail outer doublet microtubules.

Tritium-labeled viblastine, specific activity 107 Ci/mol, was prepared by acetylation of desacetylvinblastine with [3H]acetic anhydride, and has been employed in a study of vinblastine binding to tubulin. There are two high affinity vinblastine-binding sites per mole of embryonic chick brain tubulin (KA = 3-5 X 10(5) l./mol). Binding to these sites was rapid, and relatively independent of temperature between 37 and 0degreeC. Vincristin sulfate and desacetylvinblastine sulfate, two other active vinca alkaloid derivatives, competitively inhibited the binding of vinblastine. The inhibition constant for vincristine was 1.7 X 10(-5) M; and for desacetylvinblastine, 2 X 10(-5) M. The vinblastine binding activity of tubulin decayed upon aging, but this property was not studied in detail. Vinblastine did not depolymerize stable sea urchin sperm tail outer doublet microtubules, nor did it bind to these microtubules. However, tubulin solubilized from the B subfiber of the outer doublet microtubules possessed the two high affinity binding sites (KA = 1-3 X 105 l./mol). These data suggest that vinblastine destroys microtubules in cells primarily by inhibition of microtubule polymerization, and does not directly destroy preformed microtubules.     

Characterization of Membrane-Bound Aminopeptidases from Rat Brain: Identification of the Enkephalin-Degrading Aminopeptidase

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.     

Biochemical properties,homology, and genetic variation of Drosophila “nonspecific” esterases

The biochemical properties and tissue distribution of two major, soluble "nonspecific" esterases have been studied in Drosophila melanogaster, D. pseudoobscura, and related species. The "alpha-like" activity is due to a monomer enzyme (MW congruent to 60 kd) having a nonspecific tissue distribution, which was inhibited by p-hydroxymercuribenzoate (5 X 10(-4)M) plus eserine (1 X 10(-5)M) and was relatively unstable during polyacrylamide gel electrophoresis. Electrophoretograms of this enzyme could be enhanced by treating gels with beta-mercaptoethanol before staining. This procedure allowed the identification of a new alpha-esterase (Est-4) in D. pseudoobscura. The "beta-like" esterase activity (EC 3.1.1.1) is due to a dimer (MW congruent to 120 kd) in most Drosophila species. D. melanogaster and its siblings (D. simulans and D. mauritiana) were exceptions in which this enzyme had an unusual tissue distribution (increased activity in the male reproductive system) and was a monomer (MW congruent to 60 kd). Differences in the genetic variability of these esterases are discussed and interpreted by a population expansion model rather than by differences in biochemical properties of enzyme forms.     

Physicochemical characterization and tissue distribution of multiple molecular forms of fish (Trachurus trachurus) esterases

1. Tissue-specific electrophoretic patterns of multiple molecular forms of esterases were observed in fish Trachurus trachurus. They were composed of three (in intestine) to nine (in liver) bands characterized mainly as carboxylesterases and acetylesterases. 2. Two major esterase activity bands of pI 4.60 and 4.77 were accompanied by minor ones of higher pH in tissue extracts. 3. Under optimum assay conditions, liver was the richest source of esterase activity followed by intestine, stomach, brain, red muscles, heart, white muscles and gills. 4. Esterases of 70,000 and 420,000 mol. wt were resolved by gel-filtration in liver, intestine and brain. Low size esterases prevailed in liver while the opposite was the case in brain.     

Purification and properties of esterases characteristic of adult rat brain

1. A method for the partial purification of an esterase fraction, present in the brain of the adult but not the newborn rat, is described. A 54-fold purification was achieved in three steps. 2. When subjected to starch-gel electrophoresis, the purified fraction resolved into three bands of esterase activity. Two of these bands migrate close together and faster than other esterases in the brain. These two esterases are inhibited by p-hydroxymercuribenzoate but not by di-isopropyl phosphorofluoridate. The third band is di-isopropyl phosphorofluoridate-sensitive and migrates just behind the two leading esterases. 3. After treatment with di-isopropyl phosphorofluoridate, to obviate the effects of the di-isopropyl phosphorofluoridate-sensitive esterase, the enzyme preparation hydrolyses alpha-naphthyl acetate, alpha-naphthyl propionate and alpha-naphthyl butyrate, but not cholesteryl acetate. The V(max.) for the naphthyl esters decreased with increase in chain length of the acyl group. The acetate ester is hydrolysed 34 times as fast as the butyrate and about seven times as fast as the propionate derivative. The K(m) values for these three esters, measured at pH7.2 and 37 degrees , are 2.8x10(-4)m, 3.1x10(-4)m and 7.3x10(-5)m for the acetate, propionate and butyrate derivatives respectively. 4. The Hofstee (1952) plots for the kinetic data show a single line, indicating that the two most-rapidly migrating esterases, although electrophoretically separable, are not kinetically distinguishable in the substrate ranges examined.     

Identification of gonadotropin inducible, high density lipoprotein receptors in the solubilized membranes from rat ovary

Specific receptors for high density lipoproteins (HDL3) were solubilized from membranes of rat corpus luteum using different detergents. Among the detergents tested, octyl-beta-D-glucoside (40 mM) was most effective with respect to recovery of binding activity. The receptor activity released into 105,000 X g supernatant, can be assayed directly or with the precipitate obtained after dilution of the soluble supernatant. The 125I-HDL3 binding activity in the precipitated extract was linear with time, proportional to the amount of protein in the incubation mixture and saturable with increasing concentrations of 125I-HDL3. The solubilized receptor has an equilibrium dissociation constant (Kd) of 21.2 micrograms/ml and the binding activity was insensitive to Ca+2, EDTA and NaCl. These properties are similar to the membrane associated receptor. Administration of gonadotropin induced the HDL3 receptor in the solubilized membranes, suggesting that this receptor represents the physiologic receptor in the ovary.