Adrenal Cortex Extract Research Articles

Dietary and Supplement-Based Complementary and Alternative Medicine Use in Pediatric Autism Spectrum Disorder

Previous literature has shown that complementary and alternative medicine (CAM) is steadily increasing in autism spectrum disorder (ASD). However, little data is currently available regarding its use, safety, and efficacy in children with ASD. Thus, the purpose of this study is to describe the use of supplement-based CAM therapies in children between the ages of 4 to 17 years with ASD. This population-based, cross-sectional study evaluated children with ASD regarding supplement use. A total of 210 participants were recruited from a variety of sources including educational and physical activity programs, and social media to complete a questionnaire. Primary caregivers provided information on current supplement based CAM use. Data evaluated the proportion of children that used supplement therapies, the types of supplements used, reasons for use, perceived safety, and demographic factors associated with use (e.g., income, parental education, severity of disorder). Seventy-five percent of children with ASD consumed supplements with multivitamins (77.8%), vitamin D (44.9%), omega 3 (42.5%), probiotics (36.5%), and magnesium (28.1%) as the most prevalent. Several supplements, such as adrenal cortex extract, where product safety has not yet been demonstrated, were also reported. A gluten free diet was the most common specialty diet followed amongst those with restrictions (14.8%). Health care professionals were the most frequent information source regarding supplements; however, 33% of parents reported not disclosing all their child’s supplements to their physician. In conclusion, the use of supplement therapies in children with ASD is endemic and highlights the need for further research concerning public health education surrounding safety and efficacy.

Identification of the adrenocorticotropin and ginkgolide B-regulated 90-kilodalton protein (p90) in adrenocortical cells as a serotransferrin precursor protein homolog (adrenotransferrin).

Two-dimensional electrophoresis (2D-PAGE) of metabolically labeled adrenocortical proteins, identified a series of spots at a molecular size of 90 kDa [isoelectric point (pI) 6.8-7.1; p90] that was induced by ACTH, but whose intensity was reduced in cells obtained from animals treated with an extract of Ginkgo biloba (EGb 761) and its purified component ginkgolide B (GKB). We have now identified p90. GKB (2 mg/kg x d, i.p.) was administered to rats for 8 d. Adrenocortical cells were prepared and stimulated with ACTH for 3 h. Cells obtained from saline-treated rats responded to ACTH by producing high amounts of corticosterone, an effect that was inhibited in cells obtained from GKB-treated animals. Samples were fractionated by 2D-PAGE and matrix-assisted laser desorption ionization mass spectrometry analysis of the p90 spots isolated from the gels revealed sequences sharing identity with the serotransferrin precursor protein. Further PCR screening of a rat adrenal cDNA library identified a sequence with a high degree of homology (79%) to serotransferrin precursor protein, and a lesser degree to rat transferrin (54%) and human melanotransferrin (32.8%). p90, in 2D-PAGE immunoblots, was also recognized by a monoclonal antibody raised against human 97-kDa melanotransferrin. Iron binding assays with rat adrenal cortex extracts further identified a 90-kDa melanotransferrin immunoreactive protein binding iron, suggesting that the identified protein, which we name "adrenotransferrin," may have iron-binding activity. This is the first report describing the presence of a serotransferrin precursor protein homolog belonging to the transferrin family and sharing epitopes with melanotransferrin in the adrenal, its induction by ACTH, and sensitivity to GKB.

Estrogen regulates adrenal angiotensin AT1 receptors by modulating AT1 receptor translation.

Hypertension and associated cardiovascular disease increase after menopause. Angiotensin AT(1) receptor (AT(1)R) antagonists are effective treatments, in part, by inhibiting angiotensin II (Ang II)-induced aldosterone release from the adrenal zona glomerulosa (ZG). Estrogen decreases the number of AT(1)Rs in the adrenal gland and attenuates acute Ang II-induced aldosterone release. Here, we examined the effects of 17beta-estradiol (E(2)) on AT(1)R gene regulation in the rat adrenal cortex (AC). Female rats were ovariectomized and injected with vehicle or E(2). Immunohistochemistry revealed the presence of both estrogen receptor (ER)alpha and ERbeta in the ZG, and E(2) treatment increased the intensity of their nuclear staining. Under conditions in which AT(1)R maximal binding capacity was decreased by 46%, chronic miniosmotic pump Ang II-induced aldosterone secretion was reduced by 43%. E(2) treatment had no effect on AT(1a)R and AT(1b)R mRNA levels in the AC, whereas the AT(1)R mRNA polysome distribution in sucrose gradients was shifted to lighter fractions, indicating that E(2) treatment reduces AT(1)R translation. RNA binding proteins (RBPs) in AC extracts formed complexes with the 5' leader sequence (5'LS), coding region, and the 3'-untranslated region (3'UTR); however, only the activity of 5'LS RBPs was regulated by E(2) treatment. These data suggest that E(2), acting through its receptors in the ZG, reduces AT(1)R density and Ang II-induced aldosterone release, primarily by inhibiting AT(1)R translation, possibly by blocking ribosomal scanning caused by increased steric hindrance from 5'LS RBPs. Dysregulation of this posttranscriptional mechanism may contribute to the increased incidence of cardiovascular disease associated with menopause.

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.

Abscesses due to mycobacterium abscessus linked to injection of unapproved alternative medication.

An unlicensed injectable medicine sold as adrenal cortex extract (ACE*) and distributed in the alternative medicine community led to the largest outbreak of Mycobacterium abscessus infections reported in the United States. Records from the implicated distributor from January 1, 1995, to August 18, 1996, were used to identify purchasers; purchasers and public health alerts were used to identify patients. Purchasers and patients were interviewed, and available medical records were reviewed. Vials of ACE* were tested for mycobacterial contamination, and the product was recalled by the U.S. Food and Drug Administration. ACE* had been distributed to 148 purchasers in 30 states; 87 persons with postinjection abscesses attributable to the product were identified. Patient and vial cultures contained M. abscessus identical by enzymatic and molecular typing methods. Unusual infectious agents and alternative health practices should be considered in the diagnosis of infections that do not respond to routine treatment.

From the Centers for Disease Control and Prevention. Infection with Mycobacterium abscessus associated with intramuscular injection of adrenal cortex extract--Colorado and Wyoming, 1995-1996

From the Centers for Disease Control and Prevention. Infection with Mycobacterium abscessus associated with intramuscular injection of adrenal cortex extract--Colorado and Wyoming, 1995-1996

Infection With Mycobacterium abscessus Associated With Intramuscular Injection of Adrenal Cortex Extract—Colorado and Wyoming, 1995-1996

Infection With Mycobacterium abscessus Associated With Intramuscular Injection of Adrenal Cortex Extract—Colorado and Wyoming, 1995-1996

In Vivo and in Vitro Effect of Glucocorticoids on Fibroblast Growth Factor (FGF)-2 and FGF Receptor 1 Expression

In order to clarify the physiological function of fibroblast growth factor (FGF-2) in the adrenal medulla the regulation of FGF-2 and FGF receptor 1 (FGFR1) was studied in vitro and in vivo in response to glucocorticoids. To assess the effects of glucocorticoids, in vivo extracts of adrenal medulla and adrenal cortex were analyzed by RNase protection assay and Western blot analysis. PC12 cells were chosen as a model system to study the effects of glucocorticoids in vitro. In PC12 cells, dexamethasone (DEX) was found to stimulate dramatically the expression of both FGF-2 mRNA and protein. Western blot analysis revealed that exclusively the 21-kDa FGF-2 isoform was enhanced. In contrast to the FGF-2 mRNA level FGFR1 was not affected by treatment with glucocorticoids. In vivo FGF-2 mRNA level and 21-kDa FGF-2 isoform level are significantly enhanced in the adrenal medulla 24 h after DEX injection. In vivo application of DEX leads to an increase of the medullary and cortical FGFR1 transcript levels. Glucocorticoid effects on FGF-2 expression were not found in adrenal cortex, heart, skeletal muscle, and kidney, respectively, in vivo and in L6 rat myoblasts in vitro. In addition to adrenal medullary cells glucocorticoids elevated the FGF-2 mRNA and protein level also in vivo in the brain and in vitro in immortalized Schwann cells. The present results suggest that the 21-kDa FGF-2 isoform mediates a physiological function specific for neuronal tissue which is modulated by glucocorticoids.

A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the human CYP21A2 gene

Deletion mutants in the 5' upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP-dependent expression of the gene in the transient expression system using Y-1 cells (mouse adrenocortical tumor cell line). This regulatory element (-2574 to -2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP-responsive element (CRE) and a CAAT-like sequence. The CRE-like sequence has a five-base motif (5'-TGACG-3') of the palindromic CRE consensus (TGACGTCA). Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT-like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue-specific enhancers (adrenal-specific protein factor and Ad4-like sequences) and their binding factors to express a high level of cAMP-responsive expression of the CYP21A2 gene.

A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s.

Previous studies of bovine CYP11B1 gene regulation revealed six cis-acting elements, Ad1, Ad2, Ad3, Ad4, Ad5, and Ad6, in the 5' upstream region of the gene. Ad4 site was a positive transcription element in the stimulation by cAMP. Ad4-binding protein (Ad4BP) was purified from the nuclear extract of bovine adrenal cortex using affinity latex particles conjugated with polymerized Ad4 sequences. The molecular mass of the purified Ad4BP estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa. To characterize the binding specificity of Ad4BP, oligonucleotides homologous to Ad4 sequence and AGGTCA containing sequences in the promoter regions of steroidogenic P-450s were synthesized and used for gel shift analyses as competitors. The competition experiments revealed that Ad4BP bound not only to (C/T)CAAGG(T/C)(C/T), which was originally identified as the Ad4 binding site, but also to (Pu)PuPuAGGTCA. All the steroidogenic P-450 genes examined had at least one Ad4BP binding sequence. Experiments with model sequences containing various nucleotide substitutions established that (C/T)CAAGG(T/C)CA is the strongest binding sequence for Ad4BP. The expression of Ad4BP was examined with adrenal cortex cells and several other steroidogenic and nonsteroidogenic cells. Only the steroidogenic cells, the granulosa cells of bovine ovary, and I-10 cells derived from mouse Leydig cells, expressed the binding activity to Ad4 site. The presence of Ad4 site as a common cis-acting element in the genes of all the steroidogenic P-450s and the steroidogenic tissue-specific expression of Ad4BP strongly suggests that Ad4BP is an indispensable transcription factor for the expression of all the steroidogenic P-450 genes.

Tissue-Specific Transcription of P-450(11β) Gene In Vitro1

The transcriptional activity of P-450(11 beta) gene was studied with an in vitro transcription system using nuclear extracts prepared from bovine adrenal cortex. Template plasmids were constructed with the promoter region of the bovine P-450(11 beta) gene and a G-less cassette as a reporter gene. Deletion analysis of the promoter region revealed two neighboring sites, Ad1 and Ad2, which are necessary for efficient transcription of the templates. These sites are highly conserved among bovine, mouse, and human genes. Though the Ad1 site, TGACGTGA, showed high homology to the consensus sequence of the cAMP-responsive element, TGACGTCA, with one base substitution, the Ad2 site showed no similarity to any regulatory element reported so far. Gel shift assay using synthetic nucleotides containing Ad1 and/or Ad2 indicated that three factors bound to these regions in adrenal cortex nuclear extract, two factors to Ad2 and one factor to Ad1. DNase I footprint analysis also showed the binding of factors to the Ad1 and Ad2 regions. Catenated synthetic nucleotides containing Ad1 and/or Ad2 inhibited the transcriptional activity of P-450(11 beta) gene. The P-450(11 beta) promoter expressed a stronger transcriptional activity in the nuclear extract of adrenal cortex than in that of liver. On the other hand, the promoter of alpha 1-antitrypsin gene, which is expressed only in liver, showed an opposite tissue specificity of transcription. Addition of adrenal cortex nuclear extract to liver nuclear extract activated specifically the P-450(11 beta) promoter in proportion to the quantity of adrenal cortex nuclear extract added.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of adrenocortical adenosine 3',5'-monoposphate-dependent protein kinases.

In this manuscript we describe in detail the purification and biochemical and immunological characterization of cAMP-dependent protein kinases in bovine adrenal cortex, rat adrenal gland, and isolated fasciculata cells of the rat. DEAE-cellulose chromatography of bovine adrenal cortex extract yielded two major (type I and type II) cAMP-dependent protein kinase peaks and one minor cAMP-binding peak. The minor peak (peak A) eluted at 30-80 mM NaCl and corresponded to the typical type I tetrameric structure of the holoenzyme. Peak B, eluting at 80-130 mM NaCl, comprised 10-15% of the total cAMP-binding activity and was identified as dimeric type I cAMP-binding regulatory subunit of the enzyme. Peak C (major peak) eluting at high salt (130-220 mM NaCl), was different from the typical type II holoenzyme; its mol wt was relatively low (123,000), and its cAMP-binding subunit was type I rather than type II. The native enzyme contained dimeric cAMP-binding regulatory subunit and suggested the presence of only a single catalytic subunit. Based on these results and on the reduced activation of its kinase activity by cAMP, we suggest a type I trimeric structure, R I2 C, of this enzyme. Most of the bovine adrenocortical extracts (62 of 68) did not contain type II cAMP-binding regulatory subunit of the enzyme. When present, its concentration (free or part of the holoenzyme) was less than 15% of the total cAMP-dependent protein kinases. These results were further supported by the studies with rat adrenal glands and isolated fasciculata cells derived from these glands, where only the type I cAMP receptor was found. We, therefore, conclude that in contrast to the current notion, adrenal cortex contains little, if any, enzyme containing type II cAMP-binding receptor. The predominant form of the holoenzyme contains a typical type I cAMP-binding receptor, but possesses an anomalous type II-like high salt elution pattern. We suggest that the trimeric structure of this enzyme contains a typical dimeric type I cAMP-binding subunit and a single catalytic subunit, R I2 C.

Identification of a high density lipoprotein binding protein from adrenocortical membranes

A protein band with an apparent molecular weight of 78,000 daltons has been identified in the solubilised plasma membrane extract of sheep adrenal cortex which binds HDL 3 devoid of E apolipoprotein. Following ‘Western’ blotting, and development of the nitrocellulose strips with appropriate antisera and color reagent, the same band, unlike other cortical membrane proteins or albumin, bound AI and AII apolipoproteins. Human LDL bound weakly to the same band but more strongly to another two proteins of higher molecular weight. These studies confirm the same degree of specificity of HDL 3 binding found with cultured adrenal cells and strengthen the suggested existence of a specific HDL receptor.

Tissue distribution and molecular forms of a novel pituitary protein in the rat.

A sensitive and specific radioimmunoassay (RIA) was developed for a novel pituitary protein that we recently isolated from human and porcine pituitary gland and designated 7B2. By employing this RIA, we were able to detect and assay this novel protein in different rat tissue extracts. The concentrations of 7B2 in rat anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland were 10,400 +/- 804; 6,190 +/- 908; 773 +/- 50; 697 +/- 83 and 1,368 +/- 116 pg/mg tissue (wet weight, n = 10, mean +/- SEM), respectively. However, the concentrations of 7B2 were lower than 30 pg/mg tissue in extracts of pancreas, ileum and colon, and were below the sensitivity of the RIA in extracts of liver, kidney, spleen, lung, adrenal cortex and testis. Gel permeation chromatography of extracts of anterior pituitary lobe, neurointermediate lobe, hypothalamus, adrenal medulla and thyroid gland on Sephadex G-100 revealed that most of the immunoreactive (Ir)-7B2 has an apparent molecular weight of 45,000-50,000. Subsequent dissociation of this Ir-7B2 by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS) yielded an Ir-7B2 with an apparent molecular weight of around 19,000. In addition, high K+ concentration (50 mM) induced the release of Ir-7B2 from cultured cells of both rat anterior pituitary and neurointermediate lobe. Finally, Ir-7B2 was detected in the neurosecretory granule fraction prepared from porcine neurointermediate lobe. These results indicate that 7B2 may be a novel secretory protein in the pituitary gland.

Cardiovascular failure in experimental adrenal insufficiency: a historical revival.

CARDIOVASCULAR FAILURE IN EXPERIMENTAL ADRENAL INSUFFICIENCY: A HISTORICAL REVIVAL R. A. CLEGHORN* The rapid advances in cardiovascular studies in the past 4 decades by themselves have inevitably left many earlier observations stranded like flotsam after a high tide. Perhaps an equal threat to historical continuity may have arisen from the current tendency toward computer search of the literature in which, not merely does much of science appear to have been performed within the last decade or decade and a half, but anything which is not appropriately indexed according to the computer is simply lost. A purpose in the present writing is the conviction that much of the past work, buried apparently beyond the reach of memory, may well have relevance if excavated from tbe dry sands ofjournals where it lies buried. On the chance that studies with which I and others were concerned in the thirties and early forties fall into this category, I have tried to rescue and bring together some of the material which might be of interest to current pundits for heuristic as well as historical reasons. My initial involvement with studies on failing circulation, which turned out to be the core ofthe adrenal insufficiency problem, had to do with physiological investigations concerning the respiratory quotient (R.Q.) and acid-base balance in eviscerated, decerebrate cats in 1930 [1, 2]. The objective was to establish the R.Q. of this muscle preparation. It proved to be about 0.8 prior to depletion of the alkali reserve and a breakdown in circulatory homeostasis. In the following 2 years, my efforts were occupied witb the methods which had just become available for preparing an extract of the adrenal cortex and demonstrating its efficacy by maintaining the life of adrenalectomized cats [3]. The aim The author is indebted to Prof. John Grayson, Department of Physiology, University of Toronto, for his helpful criticisms and suggestions. * Emeritus professor of psychiatry, McGiIl University; consultant, Department of Psychiatry, Sunnybrook Medical Centre, 2075 Bayview Avenue, Toronto, Ontario, Canada M4H 3M5.© 1983 by The University of Chicago. AU rights reserved. 0031-5982/84/2701-0357$01.00 Perspectives in Biology and Medicine, 27, 1 ¦ Autumn 1983 | 135 was to ascertain the nature of the life-maintaining functions of the adrenal gland which were not known then, though it seemed that death occurred with a decline in the circulation. Concurrent experiments on the influence ofautonomic nerve activity on carbohydrate metabolism in the Aberdeen University laboratory where I worked seemed pertinent. This study, which was reported by Donhoffer and Macleod [4], revealed a role for an adrenal cortex hormone in the autonomic nervous system control of glycogen and sugar release from the liver. Role of the Adrenal Gland ADRENALECTOMY IN THE FROG AND EFFECT OF ADRENAL CORTEX EXTRACTS As a means of investigating the possible relationship between the adrenal cortex and autonomic nervous system, I studied adrenalectomy in frogs, repeating the 1914 experiments of Loewi and Gettwert [5], first, while in the laboratory of the Department of Medicine at the Elizabeth University in Pecs, Hungary, in 1932, and then at the Wilhelmina Gasthuis in Amsterdam (unpublished observations). I found, as had Loewi and Gettwert, that frogs subject to cauterizing of the homologue of the adrenal cortical tissue that lay on the surface of the kidney died after a few days (3-8), showing a terminal bradycardia. At death the heart was in diastole and most of the blood seemed to be in the abdominal vessels at postmortem. Oedema of the peritoneal cavity was frequent due to kidney damage while cauterizing the adrenal tissue. In experiments in which electrical stimulation was applied from head to foot of the adrenalectomized frog, the bradycardia of the adrenal insufficient state appeared sooner. Atropine placed on the heart caused a temporary quickening, but cutting of the vagi had no apparent effect on the downhill course. This suggested that stimulation of the cardioaccelerator (ca.) nerves and their exbaustion was of greater importance than vagal excitation in the development of the bradycardia. In other experiments the isolated frog heart was studied using a Straub cannula perfused with Ringer's solution. It was found that the adrenal cortical extract had a positive inotropic and chronotropic effect...

Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP)

Dispersed cells of the bovine adrenal medulla express immunoreactive vasoactive intestinal polypeptide (VIP) after 24 hours in culture, although VIP could not be detected in extracts of bovine adrenal medulla or cortex. Immunoreactive VIP eluted from a reversed-phrase chromatography column with the same retention time as authentic porcine VIP 1–28. VIP in chromaffin cells in culture appears to be contained in a secretory granule pool, since it, like methionine-enkephalin (met-enk) was released into the medium after exposure of cells to nicotine, carbachol, veratridine and elevated potassium in a dose-dependent manner. Doseresponse curves for VIP and enkephalin release by the above secretagogues were similar but not identical. Enkephalins and VIP may either be contained in separate subpopulations of chromaffin cells or co-stored in the same cells.

Molecular forms of the putative enkephalin precursor BAM-12P in bovine adrenal, pituitary, and hypothalamus.

A highly specific radioimmunoassay for one of the putative adrenomedullary [Met]enkephalin precursors, BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-OH), has been developed. The BAM-12P antibodies recognize the COOH-terminal fragment of the peptide from Arg7 to Glu12 and do not crossreact with [Met5]- or [Leu5]enkephalin or any of their COOH-terminal lysine or arginine extended analogs. Specificity for the COOH-terminal Glu-OH is suggested by the 100% crossreactivity with BAM-12P5-12 and 0.3% crossreactivity with BAM-12P5-12 amide. Using these antibodies, we have measured three forms of BAM-12P-like immunoreactivity in extracts of bovine adrenal medulla, of which the major form (greater than 90%) corresponds to BAM-12P by molecular weight. Extracts of bovine adrenal cortex contain 1% the amount of a BAM-12P-like material (Mr approximately 1400; 20 ng per gland), possibly due to crosscontamination with adrenomedullary tissue. The major form of BAM-12P-like material in extracts of bovine neurointermediate pituitaries is of higher molecular weight than authentic BAM-12P (Mr approximately 4000); the remaining material (10%) corresponds to BAM-12P by molecular weight. There is no detectable BAM-12P-like immunoreactivity in crude or purified extracts of bovine anterior pituitaries. Extracts of bovine hypothalamic tissues contain small amounts of BAM-12P immunoreactivity (approximately 2 ng per fragment) which can be detected as one molecular form corresponding to a 1400-dalton molecule. The results indicate that the enkephalin precursor found in the adrenal medulla also may be present in the pituitary and hypothalamus. Furthermore, the processing of this molecule appears to be tissue-specific.

Factors affecting the binding of [3H]adenosine 3′:5′-cyclic monophosphate to protein kinase from bovine adrenal cortex

Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.

Photoaffinity Labeling with Cyclic Nucleotides

Abstract Upon irradiation with ultraviolet light radioactivity from labeled cyclic adenosine 3' : 5'-monophosphate was incorporated into high affinity macromolecular receptors in extracts of testis and adrenal cortex. The bond formed was stable in both 10 % trichloroacetic acid and 1 % sodium dodecyl sulfate, and is consequently thought to be covalent. This incorporation was linearly proportional to the amount of cyclic adenosine 3' : 5'-monophosphate bound by these extracts. Several proteins lacking the ability to bind the cyclic nucleotide did not incorporate significant radioactivity upon irradiation. Incorporation was inhibited by unlabeled cyclic adenosine 3' : 5'-monophosphate and by analogues which compete for its binding sites. Radioactivity from tritiated cyclic guanosine 3' : 5'-monophosphate was similarly incorporated into testicular extracts upon irradiation. These observations indicate that covalent labeling of receptors for cyclic nucleotides can be accomplished by ultraviolet irradiation in the presence of the unmodified ligand.

Seasonal variations of potency of adrenal cortex extract.

Seasonal variations of potency of adrenal cortex extract.