Influence of bilateral cryptorchidism on rat pituitary luteinizing hormone charge microheterogeneity

Chromatofocusing was used to characterize the isohormones of rat luteinizing hormone (rLH) in extracts of pituitaries from intact and bilaterally cryptorchid animals. Pituitary extracts contained at least seven isoelectric variants (isohormones) or rLH: one eluted in the column void volume (Isohormone, I, pI greater than 9.8), five exhibited apparent pI's in the range of 9.25 to 8.97 (designated as Isohormones II-VI) and one bound to the column but eluted with 1.0 M NaCl (Isohormone VII, pI less than 7.0). In both intact and cryptorchid rats, a large percentage of the rLH was present as Isohormone I. Furthermore, Isohormone I was present in greater absolute and relative amounts in cryptorchid rats. All seven rLH isohormones exhibited activity in an in vitro bioassay and the biological to immunological assay (B:I) ratios decreased with their apparent pI's but were not significantly affected by cryptorchidism. When the data were expressed as the product of the amount of rLH and the B:I ratio (termed Bio-Index), Isohormone I was the predominant form of rLH in both cryptorchid and intact rats, with the Bio-Index of Isohormone I being significantly greater for cryptorchid rats. These results suggest that cryptorchidism alters the pattern of rLH isohormones in the pituitary, yielding a greater percentage of the most basic rLH isohormone, which also has the highest B:I ratio. Thus, cryptorchidism significantly alters the qualitative pattern as well as the quantity of rLH in the pituitary.     

Ovine luteinizing hormone. I. Effects of castration and steroid administration on the charge heterogeneity of pituitary luteinizing hormone

Chromatofocusing was used to separate and characterize the isohormones of ovine luteinizing hormone (oLH) in the pituitaries of rams, wethers, and wethers receiving Silastic implants containing 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2), or DHT plus E2. Extracts of anterior pituitaries were prepared by homogenization and centrifugation at 100,000 X g. Castration reduced the amount of oLH in the pituitary, even though peripheral levels were elevated. Pituitary oLH concentrations in wethers were further reduced by all three steroid treatments. When subjected to chromatofocusing on pH 10.5 to 7.0 gradients, pituitary extracts yielded eight peaks of immunoreactive oLH, which eluted with apparent isoelectric points of greater than 9.8, 9.26, 9.14, 9.07, 8.98, 8.91, and less than or equal to 7.0. These isohormones were designated A-G and Z, respectively. In rams, isohormones F and G were the predominant species, representing approximately equal to 57% of the immunoreactive oLH recovered from the column. Castration resulted in a subtle shift toward more basic isohormones. DHT administration caused an increase in the relative amount of isohormone A, whereas E2 treatment resulted in an increase in isohormone Z. DHT and E2 in combination produced increases in the relative amounts of both isohormones A and Z. All eight oLH isohormones were active in an in vitro LH bioassay and exhibited biological-to-immunological-assay (B/I) ratios in the ram ranging from 0.4 to 2.8. Isohormone F exhibited the highest B/I ratio in all the treatment groups. Similarly, isohormone F was clearly the predominant biologically active form of oLH in all groups. These results demonstrate that at least eight immunoreactive and biologically active forms of pituitary oLH can be separated by using chromatofocusing; the pattern of oLH isohormones is markedly different from that in the rat; castration has a minimal effect on the pattern of oLH isohormones in pituitary extracts; and exogenous gonadal steroid administration reduces the amount of oLH in the pituitary and changes the pattern of oLH isohormones, resulting in a higher percentage of less biologically active forms.     

Charge microheterogeneity of ovine follicle-stimulating hormone in rams and steroid-treated wethers

Chromatofocusing was utilized to separate the isohormones of ovine follicle-stimulating hormone (oFSH) in pituitary extracts from rams, wethers, and wethers receiving Silastic implants of dihydrotestosterone (DHT), 17 beta-estradiol (E2), or both DHT and E2. Pituitary extracts were prepared by homogenization and centrifugation at 100,000 x g. Extracts of ram pituitaries yielded at least nine species (isohormones) of immunoreactive oFSH having apparent isoelectric points (pIs) of greater than 7.40, 6.74, 6.52, 5.76, 5.20, 4.74, 4.44, 4.10, and less than 4.00. These isohormones were designated by the letters A-H and Z, respectively. Eighty-four percent of the immunoreactive ram oFSH recovered from the chromatofocusing column was very acidic in nature, having apparent pIs less than 5.30. The majority of the immunoreactive oFSH was focused in an area different from that of most of the immunoreactive ovine luteinizing hormone (oLH) and ovine thyroid-stimulating hormone (oTSH). Pituitary extracts from control and steroid-treated wethers also contained these nine oFSH isohormones, but significant differences were noted in the relative distribution among the isohormones. Castration resulted in a 4-fold increase in isohormone B and a concomitant reduction in isohormones E and H. DHT administration returned these levels to the values observed in the ram, whereas E2 administration produced a significant 2-fold increase in the most acidic form (isohormone Z). The implant combination produced isohormone profiles comparable to that of E2 alone. Neuraminidase treatment in vitro of both crude pituitary extracts or highly purified iodinated oFSH abolished the most acidic form and caused a marked shift in the isohormone pattern to more basic species. These results demonstrate that 1) at least nine isohormones of oFSH can be separated reproducibly from the male ovine pituitary by chromatofocusing, 2) the majority of FSH in the pituitary exists in acidic form, 3) castration and steroid administration alter the distribution of oFSH in the pituitary among its isohormones, and 4) at least a portion of oFSH-charge microheterogeneity appears to be due to the presence of sialic acid residues on the molecule.     

Charge properties of human pituitary and amniotic fluid prolactins.

The apparent isoelectric points (pI) in isoelectric focusing (IF) of human pituitary and amniotic fluid prolactin (hPRL), both non-iodinated and iodinated, were determined. Unresolved mixtures of pituitary hPRL isohormones E and F, and of at least five isohormones found in amniotic fluid, and plasma hPRL exhibit an average pI value of 6.5 - 6.7. Transient state pH values observed or previously reported for hPRL components range from pH 5.9 to 6.8 after correction to standard conditions. At pH 8.1, the major isohormone, hPRL-F, carriers a charge of 2.2 net protons per molecule. The net charge differences among isohormones E, F and G are compatible with acquisition or loss of single charged groups per 20,000 molecular weight. This net charge is similar to that of the least prolactin-bioactive major isohormone of human growth hormone (hGH-B), while the hGH with a bioactivity comparable to that of hPRL exhibits a net charge of 3.4 valence units. The "large" isohormones J and H increased net charges, by a factor of 2-3, in direct proportion to their size increments.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Ovine luteinizing hormone. III. Relationships between the charge heterogeneity of partially purified subunits and the native hormone

Extracts of anterior pituitaries from wethers were prepared by homogenization and centrifugation at 100,000 X g. When chromatofocused on pH 10.5-7.0 gradients, eight peaks of immunoreactive ovine luteinizing hormone (oLH) were observed: six exhibited apparent pIs in the range of 9.33-8.83, one eluted unbound (apparent pI greater than 9.8), and one was bound to the column (apparent pI less than or equal to 7.0). A portion of the same extracts was subjected to gel filtration on Sephadex G-100 Superfine to resolve native oLH and its uncombined subunits. oLH, oLH alpha, and oLH beta were present at concentrations of 0.907 +/- 0.127, 0.089 +/- 0.020, and 0.010 +/- 0.023 microgram/mg tissue, respectively, which translated to oLH alpha/oLH and oLH beta/oLH molar ratios of approximately equal to 0.19 and approximately equal to 0.02. Fractions containing immunoreactive oLH or uncombined subunits (oLH alpha and oLH beta) were pooled, lyophilized, and chromatofocused. Native oLH resolved from uncombined subunits by gel filtration displayed a similar pattern of isohormones to those in crude extracts. In contrast, three purified oLH preparations exhibited distinct chromatofocusing patterns. Uncombined oLH alpha in pituitary extracts resolved from native oLH by gel filtration exhibited a higher percentage (approximately equal to 37%) of acidic components when chromatofocused, while more than 97% of purified oLH alpha focused as basic forms having pIs greater than 8.9. When uncombined oLH beta in pituitary extracts was chromatofocused, more than half of the immunoreactivity was bound to the column (apparent pI less than or equal to 7.0); purified oLH beta displayed a nearly identical pattern. These results suggest that native oLH resolved from uncombined subunits by gel filtration displays a similar chromatofocusing profile to that of oLH in crude pituitary extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of human growth hormone isohormones D and E in milligram amounts (II), using isoelectric focusing on polyacrylamide gel

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing on polyacrylamide gel to fractionate the digest. Separation between the two isohormones species was optimized by employing a maximally flattened pH gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophilized. Overall yields of lyophilized isohormones D and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormone concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in the rat tibia-line assay and in the specific RIA for hGH.     

Purification and characterization of camel liver glutathione S-transferase

• 1.1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose.
• 2.2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5.
• 3.3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes.
• 4.4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

     

Immunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation.

Specific anti-(lutropin receptor) antibodies were produced by immunizing rabbits with lutropin receptor purified from pseudopregnant rat ovary. The anti-receptor serum at 1:100 dilution together with anti-(rabbit gamma-globulin) serum immunoprecipitated 70% of 3H-labelled, purified lutropin receptor and 42% of 125I-chorio-gonadotropin-receptor complex. The antiserum inhibited hormone binding to rat ovarian particles. Pseudopregnant rat ovarian particles were labelled with periodate/NaB3H4 and solubilized with Triton X-100. The Triton X-100 extract was subjected to immunoprecipitation using the anti-receptor serum. When the immunoprecipitate was dissolved and analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate under reducing conditions followed by fluorography, a receptor polypeptide with an apparent Mr 95000 was detected. A receptor down-regulating dose of choriogonadotropin was injected into pseudopregnant rats and their ovaries were removed and homogenized 4 days later, and analysed for immunoprecipitable receptors as above. No receptor molecules were found. Accordingly, the lutropin receptor molecules actually disappear rather than merely become masked from hormone during homologous down-regulation.     

Soluble and Particulate Forms of Rat Catechol-O-Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.     

Acute stimulation by lutropin of mitochondrial protein synthesis in small luteal cells

A two-dimensional electrophoretic technique was used to study the effect of acute stimulation of bovine luteal cells with lutropin on protein synthesis. Cells were incubated for 30 min with [35S]methionine in the presence of stimulating levels of luteinizing hormone (lutropin), after which the proteins were analyzed by autoradiography. Lutropin or N6,2'-O-dibutyryl-adenosine 3',5'-phosphate (Bt2cAMP) induced the labelling of three proteins, referred to as proteins A, B and C. Protein A, had a molecular mass of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4 respectively. After subcellular fractionation, the three proteins were found to be markedly concentrated in the only fraction enriched in an established mitochondrial marker. Moreover, protein A was one of the major mitochondrial newly synthesized proteins. Its appearance was observed after a 5-min incubation and was prevented by 100 microM cycloheximide. The acute accumulation of proteins A, B and C in mitochondria, the site of the rate-limiting step of steroidogenesis, suggest that they could be involved in the mechanism of stimulation by lutropin of progesterone synthesis.     

Modulation of testicular lutropin receptors in the developing male rat

In the developing male rat around 40 days of age, the testis appears to contain the maximum amount of lutropin receptors per unit weight. During this period, circulating levels of testosterone markedly increase without the concomitant major surges in lutropin levels. The increased sensitivity and responsiveness of tests to basal levels of circulating lutropin during this period is accompanied by enhanced serum prolactin levels suggesting that this hormone may be involved in this process. The finding that prolactin treatment of pubertal rats for 3 days induced the formation of more testicular lutropin receptors supports the above premise. However, shortterm immunoneutralisation of endogenous prolactin did not significantly alter the specific binding of [ 125 I ]-labelled lutropin to testicular membranes. Interestingly, during development, a close correction exists between receptor occupancy and capacity of the tissue to bind labelled lutropin. The apparent dissociation between serum lutropin levels, on the one hand and tissue occupancy and free receptor contents on the other, suggests that factors other than lutropin (presumably prolactin) are involved in the modulation of the sensitivity and the responsiveness of the testis to lutropin during early development.     

Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing

Chromatofocusing has been used as an analytical tool to check preparations of the enzyme ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) purified in either the presence or absence of the serine protease inhibitor phenylmethylsulfonyl fluoride from the cyanobacterium Anabaena sp. strain 7119. Only one isoelectric species was found when the crude extract was processed in the presence of the protease inhibitor. Nevertheless, when the inhibitor was omitted, four ionic forms of the enzyme--showing apparent pI's in the range 4.3-4.6--were separated after chromatofocusing of the purified preparation. These forms were found to differ in their specific activities, exhibiting, on the other hand, lower values than the single one obtained in the presence of the protease inhibitor. Analysis by acrylamide gel electrophoresis revealed virtually a single main protein band except for the ionic form of pI 4.39, which was clearly resolved into two active components. Except for the more basic form, which seems to be an homodimer of Mr 80,000, all the protein components were found to be monomeric species in the range Mr 33,000-38,000. These results indicate that the molecular heterogeneity of the ferredoxin-NADP+ oxidoreductase purified from the cyanobacterium Anabaena sp. strain 7119 may result from the activity of a protease present in the whole cell homogenates. On the other hand, these data also point out that chromatofocusing should be considered as an effective technique in the isolation and characterization of the different molecular forms of this enzyme.     

Biochemical characterization of mouse vitamin D-dependent calcium-binding protein. Evidence for its presence in embryonic life.

We compared immunochemical and biochemical properties of the vitamin D-dependent Ca2+-binding protein (CaBP) from rat and mouse intestine. The two intestinal CaBP species were extensively purified by gel filtration and successive anion-exchange chromatographies. Both had a similar mol.wt. of 9000. Their pI values differed markedly, being 8.0 and 4.9 in rat and mouse CaBP respectively. Accordingly, mouse CaBP displayed more anodal migration in electrophoresis under non-denaturing conditions. Both mouse and rat CaBP only exhibited partial immunochemical similarities, but their amino acid compositions were very similar. Chromatofocusing was also found to be a good method of detecting calcium-dependent changes in their pI. We developed a sensitive radioimmunoassay for mouse CaBP enabling us to detect substantial amounts of CaBP in uterus, yolk sac and chorio-allantoic placenta. During normal mouse gestation, CaBP appeared on day 12 in the chorio-allantoic placenta but was already present on day 9 in the yolk sac, where its level rose sharply between days 9.5 and 10. CaBP may therefore be considered as a new marker for mouse yolk-sac differentiation.     

Multiple sub-unit structure and microheterogeneity of α-galactosidase I from vicia faba seeds

Tetrameric α-galactosidase I from Vicia faba seeds is dissociated with urea (2.5-5.0 M) into active sub-unit forms. At low urea concentrations dissociation is only apparent if methyl α-D-mannoside is present which may be indicative of the involvement of lectin interactions in subunit aggregation. Chromatofocusing of α-galactosidase I yields multiple tetrameric forms with pI values ranging from 8.75 to 7.35. It is suggested that native α-galactosidase I is a closely related mixture of tetramers resulting from post-translational changes in the enzyme protein.     

beta-RTX. A receptor-active protein from Russell's viper (Vipera russelli russelli) venom

A single chain polypeptide, termed beta-RTX, with an apparent Mr = 9600 has been isolated from the venom of Vipera russelli russelli. It was purified by cation exchange chromatography, followed by preparative isoelectric focusing and chromatofocusing. Purity was confirmed by gel filtration, high performance liquid chromatography, gel electrophoresis, and analytical ultracentrifugation. Amino acid analysis revealed the presence of eight half-cystines, one being located at the NH2 terminus, which are linked to form four intramolecular disulfide bridges. Chromatofocusing revealed some microheterogeneity yielding three isoforms with pI values of 9.3, 9.37, and 9.48, respectively. In its native configuration, beta-RTX was not susceptible to tryptic degradation but was readily digested after reduction and alkylation. beta-RTX possesses weak phospholipase A2 activity and competes with the binding of monoamines and opiate ligands to their respective receptors. No binding to histamine, gamma-aminobutyric acid, benzodiazepine, or muscarinic receptors was observed. In vivo, whereas 100 micrograms/kg intravenous beta-RTX seemed to be without apparent effects in the rat, 10 ng/kg beta-RTX injected intracerebroventricularly caused marked sedation, with full recovery within 3 h.     

Characterization of equine luteinizing hormone by chromatofocusing

Three equine luteinizing hormone (LH) preparations (eLH-A, -B, and -C) recently have been isolated in our laboratory and were shown to differ in average basicity (eLH-A greater than -B greater than -C). The present study further characterizes these preparations by chromatofocusing. Each of these preparations are comprised of a family of isohormones, with 5 major immunoreactive peaks in the pH range of 7 to 4 (approx. pIs = 6.6, 6.1, 5.7, 5.2, and 4.8), with varying amounts of material eluting to either side of the pH gradient. Although similar isoforms are seen in all three LH preparations, the relative proportions of different isoforms vary in a manner reflecting the average charge properties of eLH-A, -B, and -C. While eLH-A contains predominantly basic forms, eLH-C consists largely of acidic material, and eLH-B is composed mostly of isohormones with pIs intermediate to eLH-A and -C. Chromatofocusing of a crude extract from a single horse pituitary gland revealed isohormone peaks corresponding to those found in the highly purified LH preparations. Peak fractions of the various isoforms were used to generate a variety of activity ratios (LH bioactivity:LH radioimmunoassay (RIA), LH radioreceptorassay (RRA):LH RIA, LH bioactivity:LH RRA, follicle-stimulating hormone (FSH) RRA:LH RIA, and FSH RRA:LH RRA activity ratios). The LH bioactivity:LH receptor binding potency ratio showed a linear increase with increasing isohormone acidity (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel glutathione transferase in human skin homologous with class alpha glutathione transferase 2-2 in the rat.

Six forms of glutathione transferase with pI values of 4.6, 5.9, 6.8, 7.1, 8.5 and 9.9 have been isolated from the cytosol fraction of normal skin from three human subjects. The three most abundant enzymes were an acidic Class Pi transferase (pI 4.6; apparent subunit Mr 23,000), a basic Class Alpha transferase (pI 8.5; apparent subunit Mr 24,000) and an even more basic glutathione transferase of Class Alpha (pI 9.9; apparent subunit Mr 26,500). The last enzyme, which was previously unknown, accounts for 10-20% of the glutathione transferase in human skin. The novel transferase showed greater similarities with rat glutathione transferase 2-2, another Class Alpha enzyme, than with any other known transferase irrespective of species. The most striking similarities included reactions with antibodies, amino acid compositions and identical N-terminal amino acid sequences (16 residues). The close relationship between the human most basic and the rat glutathione transferase 2-2 supports the classification of the transferases previously proposed and indicates that the similarities between enzymes isolated from different species are more extensive than had been assumed previously.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.