J. Wolff

Thyroidal iodide transport: IV. The role of ion size

Abstract 1. 1. The ability of various anions to react with the iodide-transport system of sheep-thyroid slices has been investigated. Km and KH values ranged from 3–5 · 10−7 M to 2 · 10−2 M to give a series of increasing K values: TcO 4 − ⩽ ClO 4 − ReO 4 − BF 4 − SeCN − ⋍ SO 5 F − SCN − I − NO 3 − ⪡ NO 2 − OCN − ⋍ Br − . 2. 2. These were compared with the partial molal ionic volumes at infinite dilution, Φ0, which shows that this series also follows a decreasing order of Φ0 (with the exceptions of ReO4−, SO3F− and SeCN−). A linear relation exists between the pK values and the partial molal ionic volumes over the range of 25–46 ml/mole. Although the pKs decline with larger volumes, a clear-cut maximum was not observed. No similar correlation exists between the pK and other size parameters. 3. 3. All of the anions (except TcO4−, which was not tested) were shown to be competitive inhibitors of iodide transport by double reciprocal plot analysis. 4. 4. The importance of size, univalency and stage for anion transport in thyroid tissue are briefly discussed in relation to certain physical properties of the ions.

The effect of thyroxine on isolated dehydrogenases

Abstract 1. (1) Thyroxine inhibits purified or crystalline malic, glutamic, lactic, triosephosphate, yeast alcohol and yeast glucose-6-phosphate dehydrogenases. The apparent K I values are 1.10 −6 M with malic dehydrogenase, 2.10 −5 M with glutamic dehydrogenase, and 1–5.10 −5 M with the other dehydrogenases. 2. (2) Triiodothyronine, triiodothyroacetic acid, and other thyroactive congeners of thyroxine are also dehydrogenase inhibitors. Various halogenated phenols ( e.g. pentabromphenol, apparent K I = 5.10 −6 M ) and xanthene dyes ( e.g. Rose bengal, apparent K i = 1.10 −5 M inhibit glutamic dehydrogenase. Other hormones, and a variety of uncoupling agents are not inhibitory. 3. (3) Dehydrogenase inhibition by thyroxine appears to be intermediate between competitive and noncompetitive in type vs. both substrates and the pyridine nucleotides. The inhibition is reversible. 4. (4) Attempts to demonstrate a thyroxine interaction with specific functional groups of the enzymes were unsuccessful. Although all dehydrogenases found here to be inhibited by thyroxine are reported to contain zinc, no direct interaction of thyroxine with this metal could be obtained. Carboxypeptidase is, however, not inhibited. 5. (5) Possible implications of the above findings for the action of the hormone in vivo are briefly discussed.

Thyroidal iodide transport. II. Comparison with non-thyroid iodide-concentrating tissues.

1. 1. The iodide-concentrating mechanisms of normal sheep thyroid, mouse submaxillary, rat-mammary and rat-thyroid tumor tissue slices have been compared. 2. 2. The tissue/medium ratio of 131Iis greater for thyroid tissue than for the other tissues under identical conditions. 3. 3. The time to attain equilibrium is 80–120 min in thyroid tissues and 30–40 min in the other three tissues. 4. 4. The tissue/medium ratio of 131I of all four tissues is reduced to half the control value at 1–3 · 10−5M iodide and the association constants vary from 2.7–3.3 · 104M. The differences in tissue/medium ratio of 131I appear to be a function of the capacity of the system rather than the affinity for iodide ion. 5. 5. Iodide accumulation in all four tissues can be shown to be a function of the external K+ concentration with half maximal stimulation of the tissue/medium ratio of 131I attained at from 0.9–2.9 · 10−3M of added K+. 6. 6. The cardiac glycosides, ouabain and scilliroside, depress the tissue/medium ratio of 131I in all four tissues. Scilliroside is a more potent inhibitor than ouabain by an order of magnitude. This depression can be partially reversed by the addition of 30 mequiv./l of K+, Rb+ or Cs+ ions.

Thyroidal iodide transport VI. On a possible role for iodide-binding phospholipids

Abstract Lipids extracted from fresh beef thyroid glands were purified by alumina chromatography. Fractions designated the “head” and “tail” fractions of the lecithin peak were used in studies of anion binding. Such binding was measured as the distribution coefficient between an aqueous phase (1 mM sodium acetate buffer (pH 4.2) and 1 mM methylmercaptoimidazole) and an organic phase containing the lipid (10 % chloroform in n -heptane). The relative binding of I − , ReO 4 − , and Br − was a function of the lipid concentration. The “head” fraction had greater overall activity and was enriched in ReO 4 − -binding capacity, whereas the “tail” fraction was relatively enriched in I − -binding capacity. The binding of both anions is reversible. The ability of other univalent anions (ClO 4 − , ReO 4 − , SCN − , Br − , NO 3 − ) to displace I − from the lipid phase was in the same sequence as their displacing ability in thyroid slices, but with a smaller spread in potencies. Destruction of activity by phosphatidase A or hydrogenation and characterization by thin-layer chromatography indicate the active lipids to be unsaturated lecithins or choline plasmalogens. The inactivity of l -α-dipalmitoyl, l -α-dioleyl, as well as soy bean and calf brain, lecithins shows that anion binding is more than a trivial property of these choline phosphatides and suggests they may be involved in thyroidal anion transport or may serve as models for systems that exhibit such anion specificity.

Aminopeptidase of the ocular lens I. Metal-ion requirements and synergistic activation

Abstract A quantitative assay for aminopeptidase was developed, using the chromogenic substrate, l -leucyl-β-naphthylamide. The enzyme from beef lens was purified about 200-fold and its properties were studied. The enzyme was activated by Co 2+ , Mg 2+ , or Mn 2+ , but maximal activity was observed only in the simultaneous presence of Co 2+ and Mn 2+ , or Co 2+ and Mg 2+ . This effect was termed synergistic activation. The activation was rapid even in crude homogenates, requiring generally less than 10 min. The metal-ion effects were shown to involve activation of the enzyme rather than the substrate. Distinct species differences were noted in the susceptibility of the lens enzyme to synergistic activation, with beef, hog, and sheep lenses possessing similar properties. The enzyme was very sensitive to inhibition by heavy-metal ions, reagents for sulfhydryl groups, and chelating agents. Exhaustive treatment of the enzyme with EDTA led to irreversible loss of activity, but if the chelator was removed before this stage was reached, most of the activity could be restored by the activation with Co 2+ plus Mn 2+ . Ca 2+ , Fe 2+ or Ni 2+ activated the enzyme partially, but could not replace Co 2+ or Mn 2+ for the synergistic effect. KCN stimulated the enzyme at 5.10 −5 −5.10 −3 M concentration, even after the peptidase had been freed of contaminating metals by treatment with EDTA. Co 2+ or Mn 2+ were able to afford the enzyme considerable protection against inhibition by mercurials, whereas Mg 2+ was not. The inhibition of the peptidase by sodium p -hydroxymercuribenzoate could be reversed by cysteine, glutathione, or mercaptoethanol. It appeared probable that Co 2+ is bound through a sulfhydryl group at the active site on the enzyme. Reexamination of the purified leucine aminopeptidase from hog kidney revealed that, after treatment with EDTA, it also exhibited some ability to be synergistically activated, and to be susceptible to inhibition by mercurials.

Thyroidal iodide transport III. Comparison of iodide with anions of periodic group VIIA

Abstract The present study offers evidence that the TcO 4 − and ReO 4 − ions, like iodide, are concentrated in sheep thyroid slices by a mechanism that requires cellular integrity, metabolic energy, and K + ion. The K m values are smaller than for iodide, being about 3·10 −7 M for TcO 4− and about 1·10 −6 M for ReO 4 − as compared to about 3·10 −5 M for I − . The ions are concentrated unchanged, whereas the lower homologue MnO 4 − was reduced in this system. Mn 2+ ion is concentrated but does not appear to require cellular integrity, metabolic energy or K + , and appears thus to be concentrated by a quite different system. The transport mechanism for TcO 4 − and ReO 4 − is similar to I − not only in the requirements for cellular integrity, K + and metabolic energy but also in that it is inhibited to the same extent by “competing” ions such as ClO 4 − , SCN − and BF 4 − . Furthermore, I − , TcO 4 − and ReO 4 − , are mutually inhibitory for transport in the thyroid slices. It is concluded that most, if not all, of the I − , TcO 4 − or ReO 4 − concentrated in thyroid tissue is transported by the same mechanism.

Melittin interactions with adenylate cyclase

Melittin, a basic polypeptide from bee venom, inhibits basal and thyrotropin-stimulated adenylate cyclase of beef thyroid membranes with a Ki approximately 10 micron. Although this property resides in the basic C-terminal and not the N-terminal portion of the molecule, inhibition is due primarily to its detergent-like nature rather than charge effects. There is also a small enhancing effect of both basal and thyrotropin-stimulated adenylate cyclase of 0.3-3 micron melittin.

Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells

Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.

Restriction of cellular iodide space by mediated efflux

Abstract 1. 1. The movements of I− and other anions in Ehrlich ascites tumor cells were studied as a model to explain the restricted anion space of certain cells. 2. 2. At 38° all the anions tested (I−, Br−, ReO4−, WO42−) penetrate only one-quarter of the cell volume. For I− this value, constant up to 6 h, is independent of the anion concentration in the medium from less than 1 μM to 0.124 M and is insensitive to anions that inhibit thyroidal I− transport competitively. 3. 3. At 4° the restriction of the I− (and Br−) compartments (but not that of ReO4− or WO42−) is abolished and the I− space approaches the water content of the cell. Upon exposure of the cold-loaded cells to 38°, a net efflux of the halide is observed until the steady-state value of one-quarter of the cell volume is again attained. 4. 4. The temperature-dependent efflux of I− is sensitive to various metabolic inhibitors, but, in ascites fluid, at a rather high concentration. 5. 5. Cardiac glycosides and quinidine markedly decrease the ability of ascites cells to restrict the anion space. The same effect is produced by nucleoside triphosphates, particularly ATP, ITP and UTP. 6. 6. The behavior of I− and Na+ was compared under identical conditions. Although certain similarities exist, a number of differences were noted. 7. 7. The evidence presented is consistent with the hypothesis that the halide restriction in ascites cells is due to a metabolism-dependent efflux.

Aminopeptidase of the ocular lens II. Substrate specificity

Abstract Evidence was sought on the identity of the lens enzyme hydrolyzing l -leucyl-β-naphthylamide with the soluble kidney leucine aminopeptidase (EC 3.4.1.1). During purification, leucinamidase activity was concentrated along with leucyl-β-naphthylamidase activity. The rate of hydrolysis of l -tryptophyl-β-naphthylamide also increased slightly upon purification, while that of l -ga-glutamimyl-β-naphthylamide remained constant, and that of l -alanyl-β-naphthylamide decreased markedly. Among several species examined, the l -leucyl compound was hydrolyzed more rapidly than the l -alanyl-β-naphthylamide only by beef, hog and sheep lenses. In six others the rate of l -alanyl-β-naphthylamide hydrolysis was 2 to 3 times that of l -leucyl-β-naphthylamide. In the ungulate-lens homogenates, l -α-glutamimyl-β-naphthylamide was less readily hydrolyzed compared with the other species. Synergistic activation by two metal ions was found with all substrates examined, and this strongly indicates that the effects of the metal ions were on the enzymes rather than on the substrates. By spectrophotometric assay the purified beef-lens enzyme was found capable of hydrolyzing l -alanyl- l -leucine twice as rapidly as l -leucinamide, while l -leucylglycine and l -alanyl- l -valine were cleaved at about the same rate as the standard substrate for aminopeptidase. Examination of kidney homogenates confirmed the findings for the lens enzyme in that hydrolysis of l -tryptophyl-β-naphthylamide appeared to be associated with that of l -leucyl-β-naphthylamide. l -α-glutaminyl-β-naphthylamide may be split by the same enzyme, but l -alanyl-gb-naphthylamide hydrolysis is due to a different enzyme.