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The present study was designed to investigate the hypothesis that interleukin‐4 (IL‐4) can induce apoptosis of human endothelial cells and to study regulatory pathways of this process. Indeed, DNA ladder assay and flow cytometry study showed that IL‐4 can induce apoptosis of endothelial cells in a time‐ and dose‐dependent manner. In addition, IL‐4 markedly increased activity of caspase‐3, and inhibition of this enzyme suppressed IL‐4‐induced apoptosis in a dose‐dependent manner. These results provide the first evidence that IL‐4 can induce apoptosis of human endothelial cells. In addition, the data indicate that the caspase‐3‐dependent pathway is critically involved in this process.

15‐Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH‐GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH‐GPx is less well investigated. In this study we cloned the PH‐GPx cDNA from a mouse fibroblast cDNA library and the PH‐GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5′‐flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.

It is generally accepted that the oxidation of pentadiene structures of polyunsaturated lipids by lipoxygenase (LOX) is regio‐ and enantio‐specific, while the free radical‐mediated lipid peroxidation gives stereo‐random racemic products. It was confirmed that the oxidation of human low density lipoprotein (LDL) by 15‐LOX from rabbit reticulocytes gave phosphatidylcholine (PC) and cholesteryl ester (CE) hydroperoxides regio‐, stereo‐ and enantio‐specifically. 15‐LOX also oxidized human plasma to give specific PC and CE hydroperoxides in spite of the presence of high concentrations of antioxidants. More CE hydroperoxides were formed than PC hydroperoxides from LDL, but the reverse order was observed for plasma oxidation. The S/R ratio of the hydroperoxides decreased during long time incubation but remained significantly larger than one, while free radical‐mediated oxidation of LDL and plasma gave racemic products.

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15- and 12-hydroxyperoxyeicosatetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n-6 and n-9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11-eicosatriynoic and 5,8,11,14-eicosatetraynoic acids in contrast to soybean lipoxygenase-1 which inactivated only by 5,8,11,14-eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n-6 and n-9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n-9 lipoxygenase from thrombocytes appears to be excluded.

Arrestin (also named ‘48 kDa protein’ or ‘S-antigen’) is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473–478]. This method does not require column chromatography and yields about 2–4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen.

The lipoxygenase from reticulocytes oxygenates 15Ls‐HETE to 8‐hydroperoxy‐15‐hydroxy‐5,9,ll,13‐eicosatetraenoic acid and 5‐hydroperoxy‐15‐hydroxy‐6,8,11,13‐eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LsHETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15Ls‐HETE.

The arachidonate 15-lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV-spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.

The pure lipoxygenase from reticulocytes converts 5,15-di-HETE at 2° C to product(s) showing a characteristic UV spectrum with maxima of strong absorbance at 300 and 316 nm and shoulders at 285 and 350 nm. Their formation was completely prevented by the lipoxygenase inhibitors, ETYA and NDGA, by heaing of the enzyme and by anaerobiosis. At 35° C the products were formed only initially and in small amounts. The reaction products were purified by SP-HPLC and shown to migrate in the region of tri-HETEs in thin-layer chromatograms. The lipoxygenase from soybeans also converts 5,15-di-HETE to these product(s) with a comparable initial rate but different kinetics. These data suggest that 5,15-di-HETE is converted via a lipoxygenase reaction to 5,6,15-trihydroxy-7,9,11,13-(e,e,c,e)- and/or 5,14,15-trihydroxy-6,8,10,12-(e,c,e,e)-eicosatetraenoic acid, both of which contain a conjugated tetraene system.

The germination process of oilseed plants is characterized by a mobilization of the storage lipids which constitute the major carbon source for the growing seedling. Despite the physiological importance of the lipid mobilization, the mechanism of this process is not well understood. Recently, it was found that a specific linoleate 13‐lipoxygenase is induced during the stage of lipid mobilization in various oilseed plants and that this enzyme is translocated to the membranes of the lipid storage organelles, the so called lipid bodies. Lipoxygenase expression was paralleled by the occurrence of enantiospecific hydro(pero)xy polyenoic fatty acid derivatives in the storage lipids suggesting the in vivo action of the enzyme. Furthermore, it was reported that oxygenated polyenoic fatty acids, in particular as 13(S)‐hydro(pero)xy‐9(Z),11(E)‐octadecanoic acid [13(S)‐H(P)ODE], are cleaved preferentially from the storage lipids when compared with their non‐oxygenated linoleate residues. These findings may suggest that 13(S)‐H(P)ODE may constitute the endogenous substrate for β‐oxidation during lipid mobilization of oilseed plants.

Reticulocyte‐type 15‐lipoxygenase is known to dioxygenate phospholipids without preceding action of phospholipases A2(PLA2). Therefore we studied the reaction of the secretory PLA2s (sPLA2) from pancreas and snake venom, and of the human cytosolic PLA2(cPLA2) with 1‐palmitoyl‐2‐arachidonoyl phosphatidylcholine (PAPC) and their 15‐lipoxygenated species (PAPC‐OOH and PAPC‐OH) either alone or as equimolar mixtures. These PLA2s cleaved PAPC‐O(O)H with higher (sPLA2) or similar rates (cPLA2) as compared with native PAPC. In mixtures, however, PAPC proved to be the preferred, albeit not exclusive substrate for all three PLA2s. Thus, partial 15‐lipoxygenation of phospholipids may also trigger liberation of arachidonic acid.

From a comparison of 9Ds‐HPODE and 13Ls‐HPODE and their methyl esters as substrates and inactivating agents of reticulocyte lipoxygenase it is concluded that the compounds inactivate the enzyme independently of any hydroperoxidase reaction. The protective effect of 4‐nitrocatechol indicates the formation of Fe(III) complexes of the enzyme with the hydroperoxyfatty acid compounds prior to inactivation.

Arrestin (also named ‘48 kDa protein’ or ‘S‐antigen’) is a soluble protein involved in controlling light‐dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473–478]. This method does not require column chromatography and yields about 2–4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S‐antigen.

Growth hormone (GH) increases plasma T 3and decreases plasma T 4in 18-day old chicken embryos, in newly hatched chicks and in adult chickens within 2 h after injection. The in vivo increase in T 3can be linked to an increased in vitro T 3recovery from liver homogenates incubated with T 4. Specific type I and type III deiodinase tests (5′D-I and 5D-III), however, show that GH has no effect at all on the amount of hepatic type I enzyme (catalyzing T 4deiodination to T 3) but acutely decreases the amount of type III enzyme (catalyzing T 3deiodination). This suggests that the GH-induced increase in plasma T 3is not due to an increased T 3production, but is the result of a decreased T 3breakdown. The lack of a stimulatory effect of GH injection in 3-day-old fed chicks might be the combined result of a low hepatic type III enzyme level and a low GH receptor availability at that stage.

© 1997 Federation of European Biochemical Societies.

The ability of the alternative pathway of complement to discriminate targets as either activators or non-activators is mediated by different binding properties of factor H to surface-associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti-H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin-generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8-15a (131X). At a molar ratio 2:1 of 125I-H:mAb all tested mAb enhanced binding of H to both activator- and non-activator-bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface-associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface-bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4-5. These results indicate that there is an additional interaction site on factor H for surface-bound C3b.

We have isolated and sequenced a fragment of 469 amino acid residues from bovine type VIII collagen. The sequence was composed of a series of Gly-X-Y repeats which was interrupted 8 times by short imperfections. The number and relative location of these interruptions were similar to those of chicken α1(X) and rabbit αl(VIII) chain triple-helical domains. Comparison to published N-terminal sequences to two triple-helical fragments of bovine type VIII collagen and to the cDNA derived sequence of the rabbit αl(VIII) chain showed that this fragment was the triple-helical domain of a second type VIII collagen chain which we designate α2(VIII).

Lipoxygenase productReticulocyte lipoxygenaseConjugated tetraeneReaction mechanism

Reticulocyte-type 15-lipoxygenase is known to dioxygenate phospholipids without preceding action of phospholipases A 2(PLA 2). Therefore we studied the reaction of the secretory PLA 2s (sPLA 2) from pancreas and snake venom, and of the human cytosolic PLA 2(cPLA 2) with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) and their 15-lipoxygenated species (PAPC-OOH and PAPC-OH) either alone or as equimolar mixtures. These PLA 2s cleaved PAPC-O(O)H with higher (sPLA 2) or similar rates (cPLA 2) as compared with native PAPC. In mixtures, however, PAPC proved to be the preferred, albeit not exclusive substrate for all three PLA 2s. Thus, partial 15-lipoxygenation of phospholipids may also trigger liberation of arachidonic acid.

The pure reticulocyte lipoxygenase converts 5,15-DiHETE via a lipoxygenase reaction to 5,14,15-trihy-droxy-6, 8,10,12-eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15-HETE and 15-HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.

We present the complete cDNA and derived amino acid sequence of the non-collagenous domain NCl of α 2(IV). Comparison with the corresponding NC1 domain of α(IV) reveals a high degree of homology at the protein level in contrast to the barely homologous triple-helical sequences of both chains.

Progesterone-binding capacity of cytochrome P450 and rate of progesterone consumption by cytochrome P450-dependent hydroxylases are highest in the Leydig cell fraction but are also detectable in the non-Leydig cell fraction of interstitial cells as well as in seminiferous tubules from rat testis. The Leydig cell compartment, however, contributes only to a minor extent to the total progesterone binding and hydroxylation within the whole testis.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site ( P4- P′ 910) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp) 5triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple-stranded cystine peptide. While the single chains showed no tendency to self-association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature ( Tm) of 33°C. This triple-helical collagen-like peptide is cleaved by the full-length human neutrophil collagenase (MMP-8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe 79-MMP-8 domain of HNC. Moreover, the full-length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP-8 for the triple-helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two α1(IV) and one α2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run-on analysis and mRNAs by RNase protection assay indicates the involvement of transcriptional as well as post-transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady-state levels α1(IV)/ α2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post-translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.

We have isolated and sequenced a fragment of 469 amino acid residues from bovine type VIII collagen. The sequence was composed of a series of Gly‐X‐Y repeats which was interrupted 8 times by short imperfections. The number and relative location of these interruptions were similar to those of chicken α1(X) and rabbit αl(VIII) chain triple‐helical domains. Comparison to published N‐terminal sequences to two triple‐helical fragments of bovine type VIII collagen and to the cDNA derived sequence of the rabbit αl(VIII) chain showed that this fragment was the triple‐helical domain of a second type VIII collagen chain which we designate α2(VIII).

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two α1(IV) and one α2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run‐on analysis and mRNAs by RNase protection assay indicates the involvement of transcriptional as well as post‐transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady‐state levels α1(IV)/ α2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post‐translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.

The arachidonate 15‐lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV‐spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.

Growth hormone (GH) increases plasma T3and decreases plasma T4in 18‐day old chicken embryos, in newly hatched chicks and in adult chickens within 2 h after injection. The in vivo increase in T3can be linked to an increased in vitro T3recovery from liver homogenates incubated with T4. Specific type I and type III deiodinase tests (5′D‐I and 5D‐III), however, show that GH has no effect at all on the amount of hepatic type I enzyme (catalyzing T4deiodination to T3) but acutely decreases the amount of type III enzyme (catalyzing T3deiodination). This suggests that the GH‐induced increase in plasma T3is not due to an increased T3production, but is the result of a decreased T3breakdown. The lack of a stimulatory effect of GH injection in 3‐day‐old fed chicks might be the combined result of a low hepatic type III enzyme level and a low GH receptor availability at that stage.

The ability of the alternative pathway of complement to discriminate targets as either activators or non‐activators is mediated by different binding properties of factor H to surface‐associated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti‐H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin‐generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8‐15a (131X). At a molar ratio 2:1 of 125I‐H:mAb all tested mAb enhanced binding of H to both activator‐ and non‐activator‐bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface‐associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface‐bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4‐5. These results indicate that there is an additional interaction site on factor H for surface‐bound C3b.

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15‐ and 12‐hydroxyperoxyeicosatetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n‐6 and n‐9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11‐eicosatriynoic and 5,8,11,14‐eicosatetraynoic acids in contrast to soybean lipoxygenase‐1 which inactivated only by 5,8,11,14‐eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n‐6 and n‐9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n‐9 lipoxygenase from thrombocytes appears to be excluded.

The lipoxygenase from reticulocytes oxygenates 15L s-HETE to 8-hydroperoxy-15-hydroxy-5,9,ll,13-eicosatetraenoic acid and 5-hydroperoxy-15-hydroxy-6,8,11,13-eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15L sHETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15L s-HETE.

From a comparison of 9D s-HPODE and 13L s-HPODE and their methyl esters as substrates and inactivating agents of reticulocyte lipoxygenase it is concluded that the compounds inactivate the enzyme independently of any hydroperoxidase reaction. The protective effect of 4-nitrocatechol indicates the formation of Fe(III) complexes of the enzyme with the hydroperoxyfatty acid compounds prior to inactivation.

The 48-kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5–10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48-kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP-binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP-binding protein thereby displaces 48-kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.

A selectively deuterated dihydrofolate reductase from L. caseihas been prepared containing partially deuterated aromatic amino acids. This provides simplified 2D NMR spectra and allows signals from all 8 Phe residues to be identified. The pattern of deuteration is such that (i) the only cross-peaks detected in the aromatic region of the 2D COSY spectrum are those between the Phe 2′,6′ and 3′,5′ protons and (ii) chemical shift degeneracy in the aromatic region is removed thus allowing unambiguous assignment of cross-peaks in 2D NOESY spectra required for specific assignment purposes.

Enzymes catalyzing the outer ring deiodination (ORD) of iodothyronines are important for the regulation of thyroid hormone bioactivity. We have studied ORD of thyroxine (T4) and 3,3',5'-triiodothyronine (rT3) in liver and kidney microsomes offish, i.e. tilapia ( Oreochromis niloticus). Tilapia kidney contains an enzyme which resembles the mammalian selenoenzyme type I iodothyronine deiodinase (ID-I) with respect to substrate preference (rT3 > T4) and high (≈ μM) Kmvalues, but is much less sensitive to selenocysteine (Sec)-targeted inhibitors, including 6-propyl-2-thiouracil (PTU). In contrast, tilapia liver contains an enzyme very similar to mammalian type II deiodinase (ID-II) with respect to substrate preference (T4 > rT3), low (≈ nM) Kmvalues, and lack of sensitivity to Sec inhibitors.

We present the complete cDNA and derived amino acid sequence of the non‐collagenous domain NCl of α2(IV). Comparison with the corresponding NC1 domain of α(IV) reveals a high degree of homology at the protein level in contrast to the barely homologous triple‐helical sequences of both chains.

The pure reticulocyte lipoxygenase converts 5,15‐DiHETE via a lipoxygenase reaction to 5,14,15‐trihy‐droxy‐6, 8,10,12‐eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15‐HETE and 15‐HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site () of type I collagen linked to a C‐terminal cystine‐knot, and N‐terminally extended with (Gly‐Pro‐Hyp)5triplets for stabilization of the triple‐helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triple‐stranded cystine peptide. While the single chains showed no tendency to self‐association into triple helices, the heterotrimer (α1α2α1′) was found to exhibit a typical collagen‐like CD spectrum at room temperature and a melting temperature (Tm) of 33°C. This triple‐helical collagen‐like peptide is cleaved by the full‐length human neutrophil collagenase (MMP‐8) at a single locus fully confirming the correct raster of the heterotrimer. Its digestion proceeds at rates markedly higher than that of a single α1′ chain. In contrast, opposite digestion rates were measured with the catalytic Phe79‐MMP‐8 domain of HNC. Moreover, the full‐length enzyme exhibits Kmvalues of 5 μM and 1 mM for the heterotrimer and the single α1′ chain, respectively, which compare well with those reported for collagen type I (∼ 1 μM), gelatine (∼ 10 μM) and for octapeptides of the cleavage sequence (≥1 mM). The high affinity of the MMP‐8 for the triple‐helical heterotrimer and the fast digestion of this collagenous peptide confirm the decisive role of the hemopexin domain in recognition and possibly, partial unfolding of collagen.

Rate constants for the subtilisin‐catalyzed proteolytic inactivation of cytochrome P450c17 (CYP17), the endoplasmic reticulum membrane‐bound limiting enzyme of gonadal androgen synthesis, have been determined in the absence and presence of various CYP17 ligands and correlated with fractional enzyme saturation (Y). Extrapolation to Y= 1 reveals 15.1‐, 4.0‐ and 7.4‐fold enzyme stabilization with progesterone (substrate‐type ligand), testosterone (product‐type ligand) and ketoconazole (imidazole‐type inhibitory ligand), respectively. Structural features of ligand accommodation can therefore be monitored by the susceptibility of target enzymes to proteolysis. It is further proposed that specific protection of a membrane protein by ligand binding during proteolytic digestion may assist in the purification of that protein. Evidence is finally presented that the gonadotropin‐induced rapid CYP17 down‐regulation is not promoted by an elevation of steroid hormone levels.

Enzymes catalyzing the outer ring deiodination (ORD) of iodothyronines are important for the regulation of thyroid hormone bioactivity. We have studied ORD of thyroxine (T4) and 3,3',5'‐triiodothyronine (rT3) in liver and kidney microsomes offish, i.e. tilapia (Oreochromis niloticus). Tilapia kidney contains an enzyme which resembles the mammalian selenoenzyme type I iodothyronine deiodinase (ID‐I) with respect to substrate preference (rT3 > T4) and high (≈ μM) Kmvalues, but is much less sensitive to selenocysteine (Sec)‐targeted inhibitors, including 6‐propyl‐2‐thiouracil (PTU). In contrast, tilapia liver contains an enzyme very similar to mammalian type II deiodinase (ID‐II) with respect to substrate preference (T4 > rT3), low (≈ nM) Kmvalues, and lack of sensitivity to Sec inhibitors.