Bovine Adrenal Glands Research Articles

Actions of somatostatin on perfused bovine adrenal glands and cultured bovine adrenal medullary cells

The effects of somatostatin on catecholamine secretion and inositol phosphate accumulation have been studied using isolated perfused bovine adrenal glands and cultured bovine adrenal medullary cells. Somatostatin had no effect on basal adrenaline or noradrenaline secretion from either preparation. At concentrations above 1 μM, somatostatin inhibited the secretion of both catecholamines induced by 5 μM nicotine from cultured chromaffin cells. In contrast, over the concentration range 0.1 nM–10 μM, somatostatin had no effect on the secretory responses produced by 10 nM angiotensin II or 1 μM histamine. Inositol phosphate accumulation in cultured bovine adrenal medullary cells was unaffected by 0.1 nM–0.1 μM somatostatin, however at 1 and 10 μM somatostatin it was significantly increased, by 2% and 103% respectively. The effects of somatostatin (0.1 nM–10 μM) and of 50 μM muscarine on inositol phosphate accumulation were simply additive. Similarly, somatostatin at 0.1 nM and 10 nM together with 10 nM angiotensin II or 1 μM histamine produced additive inositol phosphate responses. In contrast, 1 μM somatostain gave significantly more-than-additive (synergistic) inositol phosphate responses with angiotensin II and histamine. The results suggest that some adrenal medullary cells possess several types of receptors, and that these receptors may interact to produce non-additive responses.

Immunohistochemical Demonstration of Cholesterol Side-chain Cleavage Cytochrome P-450 in Bovine and Human Adrenals

Cytochrome P-450 specific for cholesterol side-chain cleavage (P-450SCC) was purified from the bovine adrenal and a specific antibody was raised in rabbits. The antiserum was applied for immunohistochemical visualization of the P-450SCC in the bovine and human adrenal cortex. The immunoreactivity was intense in the zona fasciculata (ZF) and reticularis (ZR) while weak in the zona glomerulosa (ZG) in the normal adrenals. In adrenocortical hyperplasia, a marked immunoreactivity was observed in the ZG in idiopathic hyperaldosteronism and the inner ZF and reticularis particularly in cells of micronodules in Cushing's disease, corresponding to cells with active steroidogenesis. In aldosteronoma and adenoma with Cushing's syndrome, P-450SCC was generally present in compact cells of adenomas.

Structural identification, subcellular localization and secretion of bovine adrenomedullary neuromedin C [GRP-(18–27)

Bombesin-like immunoreactivity (BLI) was purified from acid (HCl) extracts of bovine adrenal medulla. High performance liquid chromatography (HPLC) on a μ-Bondapak C18 column revealed the presence of five molecular forms of BLI, one coeluting with synthetic gastrin releasing peptide (GRP), the mammalian counterpart of amphibian bombesin, one coeluting with neuromedin C, one coeluting with neuromedin B and the two other ones coeluting with the oxidized forms of neuromedins B and C. The material corresponding to neuromedin C was purified to homogeneity and its amino acid composition and sequence corresponded to those expected for neuromedin C. HPLC analysis on an analytical SP-5PW column of subcellular extracts of bovine adrenal medulla indicated that neuromedin C is almost exclusively localized in secretory granules. The neuropeptide function of neuromedin C and/or other BLI peptides at this level was supported by the stimulatory effect of carbamylcholine (500 μM) on the release of BLI (4.5-fold increase over the basal release of 19 fmol/5 min) from perfused bovine adrenal glands.

Immunohistochemical study of cytochrome P-450 17α in human adrenocortical disorders

Cytochrome P-450 specific for steroid 17α-hydroxylation (P-450 17α) was immunolocalized in normal and hyperfunctioning adrenal glands of pigs, bovines, and humans, using a specific IgG fraction raised against the enzyme. P-450 17α was present in the zona fasciculata (ZF) and zona reticularis (ZR), but not in the zona glomerulosa (ZG), in pig, bovine, and human adrenal glands. In the adrenal glands of patients with Cushing's disease, the positive immunoreactivity to P-450 17α was intense in ZF and ZR, particularly in cortical micronodules, corresponding to the sites of active steroidogenesis. Cells of hyperplastic ZG and outer ZF in the adrenal glands of idiopathic hyperaldosteronism were negative for P-450 17α. In aldosteronoma, positive immunoreactivity was observed in some tumor cells, which is consistent with cortisol production and its responsiveness to ACTH in aldosteronoma. In the attached adrenal glands of aldosteronoma, the immunoreactive P-450 17α was clearly present in the inner ZF and ZR, suggesting persistent androgen production. In Cushing's adenoma, the positive immunoreactivity was intense in tumor cells, and the ZR of the attached adrenal glands was weakly immunoreactive.

Calcium-binding proteins in the parathyroid gland: Detailed studies of parathyroid secretory protein

Abstract In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.

3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

Localization of angiotensin II binding sites in the bovine adrenal medulla using a labelled specific antagonist

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [ 125I] — [Sar 1, Ile 8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.

Stimulation of cholinergic receptor mediated secretion from the bovine adrenal medulla by neuropeptide Y

The bovine adrenal medulla has been shown to possess binding sites for neuropeptides Y (NPY) and to release NPY in response to nicotinic receptor stimulation. Therefore we chose to investigate the influence of this peptide on adrenomedullary secretion using the retrogradely perfused bovine adrenal gland. The secretion of enkephalin-like peptides, norepinephrine and epinephrine was monitored after nicotinic cholinergic receptor stimulation in the presence and absence of NPY. NPY, alone, had no effect on secretion from the adrenal gland but produced a dose dependent increase in the secretion of enkephalin-like peptides and catecholamines when the cholinergic agonist, 1,1 dimethyl-4-phenylpiperizinium iodide, was present. The increase was significant at 1 × 10 −8M when compared to release in the absence of NPY. The stimulatory action of other cholinergic agonists (nicotine and acetylcholine) was likewise potentiated by the addition of the neuropeptide. Peptide YY and pancreatic polypeptide did not mimic the effect of NPY when examined at the same concentration. In contrast to the potentiation observed in the perfused adrenal gland, NPY (1 × 10 −8M) inhibited the cholinergic mediated release of enkephalin-like peptides and catecholamines from cultured bovine chromaffin cells. These data suggest that NPY may have the capacity to augment cholinergic receptor mediated secretion from the bovine adrenal gland.

The distribution of opioid binding subtypes in the bovine adrenal medulla

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [ 3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1–13). Specific [ 3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1–13). Delta opioid sites, labelled with [ 3H][ d-Ala 2, d-Leu 5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [ 3H][ d-Ala 2, NMePhe 4,Gly-ol 5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.

Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein.

The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland. Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography. On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points. The major peak of the substrate was further purified. It was purified about 1,800-fold with a recovery of 2.2% by the above procedures. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000. The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein. A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol. GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP. Thus, the ADP-ribosylation substrate is a GTP-binding protein. This protein, designated Gb (b for botulinum), is widely distributed in various tissues. It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.

Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant ( K d) of 2.32 ± 0.33 × 10 −10 M (mean ± SE) and a maximal binding capacity ( B max) of 36.7 ± 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANp. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC 50 values for hANP and pBNP: 8.5 × 10 −10 and 6.5 × 10 −10 M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.

Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

Potentiation of evoked adrenal catecholamine release by cyanide: Possible role of calcium

The effect of cyanide on release of catecholamines was evaluated in isolated bovine adrenal glands stimulated with 4 different agonists. Cyanide (0.1 – mM) increased catecholamine release induced by barium or cadmium 2−3-fold. Acetylcholine or potassium induced secretion of adrenal catecholamines was also enhanced by cyanide, but only to the extent of 30–50%. These data suggest that cyanide acts by multiple mechanisms to enhance evoked catecholamine release. The above results may be partly explained by the fact that cyanide inhibited 45Ca efflux from stimulated bovine adrenals. Changes in plasma membrane permeability may be crucial in the alterations of ion flux and evoked catecholamine release caused by cyanide.

1-alkyl analogues of aminoglutethimide: Comparative inhibition of cholesterol side chain cleavage and aromatase and metabolism of the 1-propyl derivative, a highly selective inhibitor of aromatase

A homologous series of 1- n-alkyl-derivatives of aminoglutethimide (AG) has been synthesised and tested for inhibitory activity towards the cholesterol side chain cleavage enzyme (desmolase) from bovine adrenals and human placental aromatase in an attempt to find a selective aromatase inhibitor. Activity against desmolase declined from an ic 50 value of 30 m̊M for the parent drug to 220 m̊M for the n-propyl derivative but increased again thereafter. Against aromatase, activity was least for the methyl and ethyl derivatives and highest ( ic 50 = 1.6 m ̊ M ) for the hexyl and octyl analogues. The optimal ratio ic 50 (desmolase) : ic 50 aromatase of 44 was found for the n-propyl derivative, which was therefore selected for preliminary metabolism studies using rat and mouse liver microsomes and hepatocytes and in these species in vivo. There were parallels with AG, most notably in the analogous formation from the n-propyl derivative of an arylhydroxylamine in the mouse.

Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal.

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

Analogues of aminoglutethimide based on 1-phenyl-3-azabicyclo[3.1.0]hexane-2,4-dione: selective inhibition of aromatase activity.

In exploring the structural features responsible for the inhibitory activity of aminoglutethimide [3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione] (1) toward the cholesterol side chain cleavage (CSCC) enzyme from bovine adrenals and the human placental aromatase enzyme, analogues have been synthesized in which the piperidine-2,6-dione ring is replaced by substituted or unsubstituted azabicyclo[3.1.0]hexane-2,4-dione rings. The unsubstituted analogue 1-(4-aminophenyl)-3-azabicyclo[3.1.0]hexane-2,4-dione (9a) is a slightly more potent inhibitor of aromatase than 1 (Ki = 1.2 microM, cf. 1.8 microM for 1) but is noninhibitory toward the CSCC enzyme. The substituted analogues 1-(4-aminophenyl)-3-butyl-3-azabicyclo[3.1.0]hexane-2,4-dione (9e) and 1-(4-aminophenyl)-3-pentyl-3-azabicyclo[3.1.0]hexane-2,4-dione (9f) are approximately 100 times more potent than 1 (Ki values of 1, 9e, and 9f are 1.8, 0.015, and 0.02 microM, respectively) in inhibiting aromatase, with no significant activity toward the CSCC enzyme. Type II difference spectra were exhibited by 1, 9a, and 9f in their interaction with the aromatase enzyme (respective Ks values of 1, 9a, and 9f are 0.13, 0.08, and 0.01 microM). Modification of the para amino function by alkylation, its relocation, replacement by H, or replacement by a methyl, aldehyde, or secondary alcohol group produced analogues that were inactive toward both enzyme systems.

Human growth hormone binds to lactogenic receptors in bovine, ovine and rat adrenals.

The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization.

Dopamine β-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone

Treatment of homogeneous dopamine β-hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper-quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O 2. DBH is the first copper-quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed ‘hydrazine method’ [(1987) FEBS Lett. 221, 299-304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.