On the binding of steroid sulfates to albumin

3H-Labeled steroid sulfates, sulfate of estrone (E1S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E1S). The apparent equilibrium constants (K) of the tracers did not measurably change at concentrations of the non-radioactive sulfates below 10(-5) mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of 3H-E1S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E1S in vivo, because of its preferential occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

In vitro amiodarone protein binding and its interaction with warfarin

Summary The binding of amiodarone to human plasma protein and to bovine serum, albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.This work was partially funded by the CNR (National Research Council, Rome, Italy), Program on Clinical Pharmacology and Rare Diseases. The authors would like to thanks Drs E. Marzi and E. riva for their help.     

Binding of centchroman—a nonsteroidal antifertility agent to human plasma proteins

Summary Centchroman, a non-steroidal antifertility agent showed a low affinity (K_d=13.19×10^–6 M) and nonsaturable binding to human plasma. Centchroman did not complete either with sex hormone binding globulin or corticosteroid binding globulin. Polyacrylamide gel electrophoresis and temperature dependent binding characteristics revealed that the protein responsible for centchroman binding to human plasma resembles albumin.Acknowledgment. The authors are grateful to Dr. Nitya Nand for his interest in this investigation. CDRI Communication No. 3333.     

NADP-isocitric dehydrogenase of gerbil adrenal mitochondria: Support of steroid hydroxylation

Summary In gerbil adrenal cortex the activity of intramitochondrial NADP-linked isocitric dehydrogenase (IDH) is up to 10-fold greater than the NAD-linked IDH. The NADP-IDH, apparent K_m 0.58 mM, V_max 280 nmoles/min/mg mitochondrial protein, appears to be the major source of reducing equivalents to support adrenal mitochondrial steroid 11B- and 19-hydroxylation in this species.     

In vitro amiodarone protein binding and its interaction with warfarin

The binding of amiodarone to human plasma protein and to bovine serum albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.     

Differential effects of prostaglandins and isoproterenol on cAMP content and Na,K pump activity in rat submandibular acini

Summary The -adrenergic agonist isoproterenol and prostaglandins E₁ and E₂ (but not F₂) increased the cAMP content of rat submandibular acini in vitro, but only isoproterenol enhanced ouabain-sensitive⁸⁶Rb (K) uptake. These findings suggest that cAMP is not involved in the activation of the Na, K pump in salivary cells.     

17β-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes

Effects of 17-estradiol (E₂) in vitro on Na-dependent Ca²⁺ efflux from, and depolarization-induced Ca²⁺ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E₂ (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca²⁺ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca²⁺ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca²⁺ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E₂, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca²⁺ transport mechanisms in nerve endings.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Effect of various solubilizers on angiotensinII receptors in bovine adrenocortical plasma membranes

Summary 3 monionic detergents, Triton X-100, Lubrol WX and NP-40, inhibited binding of [³H]-AT_II to bovine adrenocortical plasma membranes. This effect appeared to be direct and not due to solubilization of the AT_II receptor by these agents. Sodium deoxycholate and the chaotropic ions, ClO ₄ ^– and Br^–, produced effects similar to the nonionic detergents.Supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. The authors acknowledge the technical assistance of Mrs D. Michaud and the secretarial aide of Mrs D. Huot-Blais, and Miss L. Leblanc.     

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae ofDrosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm,Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5×10^–6 to 5×10^–4 M.     

Dexamethasone-induced neutrophilia. Negative correlation with increased plasma adrenaline concentrations

Summary Maximal increments in adrenaline and dexamethasone (DXM) plasma concentrations were observed c15 (T₅₀ 40 min) and 30 (T₅₀ 210–240 min) minutes after an i.v. DXM dose (6 mg/m² BSA) in man. There appears, however, to be no direct interaction between these agents in the development of induced neutrophilia, which occurs c240 min postinjection.Acknowledgments. The authors wish to thank Mrs C. Ditzler, Merck Sharp & Dohme Research Laboratories, USA, for determining the plasma dexamethasone concentrations J. M. M. is currently on sabbatical leave from McGaw Laboratories, USA. C. R. B. is a Rhodes scholar.     

The structures of minor congeners of detoxin complex,the selective antagonist of blasticidin S1

Summary The structures of the minor congeners of detoxin complex, viz., detoxins E₁, C₁, C₂, C₃, B₁, B₃ and A₁ have been established on the basis of spectral and degradative evidence.Acknowledgment. This work was supported by a Grant-in-Aid for Special Project Research (510208) from the Ministry of Education, Science and Culture, Japan. We are grateful to Kaken Chemical Co. Ltd for the supply of the detoxin complex. This is Part V of studies of Detoxin Complex, the Selective Antagonists of Blasticidin S'. For Part IV, see Ogita et al.⁸.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Ketoconazole,an inhibitor of calcium transport in skeletal muscle sarcoplasmic reticulum

Summary Ketoconazole, an antimycotic agent, inhibits calcium binding and accumulation, and induces calcium release in sarcoplasmic reticulum. The Mg²⁺-ATPase and the (Ca²⁺+Mg²⁺)-ATPase activities are stimulated at low but inhibited at high concentrations of ketoconazole.The author wishes to thank Dr K. S. Cheah for discussion and Mr C. C. Ketteridge for preparing the sarcoplasmic reticulum and carrying out the ATPase assays.     

The site of 25-hydroxycholecalciferol stimulated protein synthesis in the rat kidney

Summary Autoradiographs of the kidneys of rachitic rats dosed in vivo with 250 pmoles 25-hydroxycholecaliferol (25-OHD₃) and³H-leucine showed increased grain counts in portions of proximal renal tubules. On incubation of kidney slices the synthesis of only 1 cytosol protein was found to be stimulated by the steroid. On disc gel electrophoresis it had the characteristics of renal calcium binding protein.     

The human α2-plasmin inhibitor: functional characterization of the unique plasmin(ogen)-binding region

The human α₂-plasmin inhibitor (A2PI) possesses unique N- and C-terminal extensions that significantly influence its biological activities. The C-terminal segment, A2PIC (Asn³⁹⁸-Lys⁴⁵²), contains six lysines thought to be involved in the binding to lysine-binding sites in the kringle domains of human plasminogen, of which four (Lys⁴²², Lys⁴²⁹, Lys⁴³⁶, Lys⁴⁵²) are completely and two (Lys⁴⁰⁶, Lys⁴¹⁵) are partially conserved. Multiple Lys to Ala mutants of A2PIC were expressed in Escherichia coli and used in intrinsic fluorescence titrations with kringle domains K1, K4, K4 + 5, and K1 + 2 + 3 of human plasminogen. We were able to identify the C-terminal Lys⁴⁵² as the main binding partner in recombinant A2PIC (rA2PIC) constructs with isolated kringles. We could show a cooperative, zipper-like enhancement of the interaction between C-terminal Lys⁴⁵² and internal Lys⁴³⁶ of rA2PIC and isolated K1 + 2 + 3, whereas the other internal lysine residues contribute only to a minor extent to the binding process. Sulfated Tyr⁴⁴⁵ in the unique C-terminal segment revealed no influence on the binding affinity to kringle domains.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

A note of the dual effect of prostaglandin E1 on the responses of the guinea-pig vas deferens to nerve stimulation

Résumé La prostaglandine E₁ diminue les potentiels de jonction excitateurs du muscle lisse du vas deferens de cobaye en réponse à une stimulation nerveuse, mais provoque aussi une dépolarisation modérée de ce tissu. L'effet inhibiteur de la drogue sur la réponse motrice de cet organe à une stimulation nerveuse est attribué à la 1^e de ces actions. L'effet stimulant sur la réponse motrice parfois constaté, est attribué à la seconde action.

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The generous hospitality of ProfessorEdith Bülbring F. R. S. is gratefully acknowledged. The investigation was supported by a grant to ProfessorBülbring from the Medical Research Council.