The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin

NADH cytochrome b₅ oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b₅ (b₅) and cytochrome b₅ reductase (b₅R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b₅ domain and a construct containing the CS and b₅R domains (CS/b₅R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L³⁴MDWIRL⁴⁰ motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b₅ domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b₅ domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp¹²⁰ (corresponding to invariant Trp³⁷ in Ncb5or) to be part of an 11-residue α-helix (S¹¹⁶QMDWLKLTRT¹²⁶) packing against two of the four helices in the b₅ domain that surround heme (α2 and α5). The Trp¹²⁰ side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr⁸⁵ and Tyr⁸⁸ (α2), Cys¹²⁴ (α5), and Leu⁴⁷ in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp³⁷ in nucleating the formation of the N-terminal helix, whose location in the N/b₅ module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b₅ module as reflected by its existence in a class of cytochrome b₅ proteins from three kingdoms among eukaryotes.     

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells. The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60–70%) and lipid (40–30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid-Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre-treatment of intact erythrocytes with 4,4′-diisothiocyano-2,2′-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporation of procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.     

Potentiation of thyroxine 5-deiodination by aminotriazole

Aminotriazole, a goitrogen, in addition to its known inhibitory effects on the thyroid, demonstrated a unique effect on peripheral deiodination of thyroxine (T4). In contrast to the well-known peripheral effects of goitrogens such as propylthiouracil in inhibiting 5'-deiodinase activity, i.e., to effect a decrease in T4 to triiodothyronine (T3) conversion, aminotriazole had no effect on the 5'-deiodinative pathway. Rather, this goitrogen appeared to stimulate the alternative pathway, viz. T4 5-deiodination, resulting in an increased reverse triiodothyronine (rT3) serum concentration. This was shown in comparisons of serum T4, T3 and rT3 concentrations and serum T3/T4 and rT3/T4 ratios between rats treated with aminotriazole and T4, and rats treated with T4 alone. The finding that aminotriazole may specifically enhance T4 5-deiodination, independently of T4 5'-deiodination, is novel, as this has not been observed in the case of other goitrogens. It is of interest that this goitrogen is devoid of sulphur, which is a prominent constituent of thiourylene compounds which have been noted to affect 5'-deiodination. The potentiating effect of aminotriazole on 5-deiodination of T4 was not attributable to dietary factors.     

Apparent increase in type I 5'-deiodinase activity induced by antiepileptic medication in mentally retarded subjects

BACKGROUND/OBJECTIVES: Thyroid function measurements in 3 mentally retarded patients treated with antiepileptic drugs (phenytoin or carbamazepine) showed normal thyroid-stimulating hormone (TSH) responses in spite of markedly low levels of total thyroxine (T(4)), triiodothyronine (T(3)), and free thyroxine (FT(4)) concentrations; free triiodothyronine (FT(3)), as well as mean thyroxine-binding globulin (TBG) concentrations were normal. The objective of the present investigations was to determine if antiepileptic medication in these patients contributed to the disparate TSH and thyroid hormone (TH) levels. METHODS: Thyroid tests and other laboratory parameters were measured by conventional techniques. RESULTS: Circulating TH changes noted in retarded patients were similar to those observed in control subjects receiving carbamazepine alone. Reverse T(3) (rT(3)) levels in all patients were either undetectable or below the normal range. CONCLUSIONS: As type I 5'-deiodinase has a higher affinity for rT(3) than T(4), an increased activity of this enzyme would enhance rT(3) deiodination and reduce serum rT(3) concentration whereas enhanced T(4) deiodination would aid in normalizing intracellular FT(3) concentration. The finding of normal serum FT(3) concentration was consistent with normal TSH response and clinical euthyroidism in both retarded and control subjects. While phenytoin-induced increase in type I 5'-deiodinase has been previously noted, the present studies demonstrate a similar effect of carbamazepine on 5'-deiodinase.     

Lowering of T3 and rise in reverse T3 induced by hyperglucagonemia: altered thyroid hormone metabolism, not altered release of thyroid hormones

Recently we reported that hyperglucagonemia induced by glucagon infusion causes a decline in serum T3 and a rise in reverse T3 in euthyroid healthy volunteers. These changes in T3 and rT3 levels were attributed to altered T4 metabolism in peripheral tissues. However, the contribution of altered release of thyroid hormones by the thyroid gland could not be excluded. Since the release of thyroid hormones is inhibited in primary hypothyroidism and is almost totally suppressed following L-thyroxine replacement therapy, we studied thyroid hormone levels for up to 6 hours after intravenous administration of glucagon in subjects with primary hypothyroidism who were rendered euthyroid by appropriate L-thyroxine replacement therapy for several years. A control study was conducted using normal saline infusion. Plasma glucose rose promptly following glucagon administration demonstrating its physiologic effect. Serum T4, Free T4, and T3 resin uptake were not altered during both studies. Glucagon infusion induced a significant decline in serum T3 (P less than 0.05) and a marked rise in rT3 (P less than 0.05) whereas saline administration caused no alterations in T3 or rT3 levels. Thus the changes in T3 and rT3 were significantly different during glucagon study when compared to saline infusion. (P less than 0.01 for both comparisons). Since, the release of thyroid hormones is suppressed by exogenous LT4 administration in these subjects; we conclude that changes in serum T3 and rT3 observed following glucagon administration reflect altered thyroid hormone metabolism in peripheral tissues and not altered release by the thyroid gland.     

Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells

A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. The results indicate that (a) the native neonatal RBC membranes contain higher levels of membrane-bound hemoglobin (MBHb) than the adult RBC membranes. (b) The content of MBHb increases when RBCs are incubated with increasing concentrations of hydrogen peroxide (H2O2), more so in neonatal than in adult RBCs; however, neonatal RBC membrane proteins are less susceptible to H2O2 oxidation than adult ones. This could be attributed to the fact that Hb F, which is more susceptible to oxidation than Hb A, adds to the reduction potential of neonatal RBC (in which it is present in large amounts) and partially protects neonatal membrane proteins against oxidant stress compared to Hb A in adult RBC. (c) In both neonatal and adult RBCs, Spectrin 1 is relatively more susceptible to oxidant stress than spectrin 2, and spectrins in adult RBC are more labile for peroxidation than the spectrins in neonatal RBC. (d) Based on electrophoretic studies with and without reduction of membranes with mercaptoethanol, we have classified two types of MBHb: Type I is adsorbed to membrane by noncovalent interactions and Type II MBHb is chemically crosslinked to membrane components by disulfide bridges; the content of both these types increases when RBCs are incubated with increasing concentrations of H2O2. (e) Band 6 protein is present in higher amounts in neonatal than in adult RBC membranes. (f) Since the total content of MBHb increases linearly with the level of oxidant stress, we suggest that it could be used as a marker for oxygen radical-induced injury to tissues.     

Circulating reverse triiodothyronine (rT3) in the dog

Plasma reverse triiodothyronine (rT₃) concentration was measured by radio immunoassay (RIA) in a group of 15 dogs. The mean rT₃ concentration was 187 ng/100 ml which was 3 times higher than radioimmunoassayable triiodothyronine (T₃) concentration. rT₃ measurement in thyroid and peripheral venous plasma in 3 dogs showed that the unusually high circulating rT₃ levels in this species could not be explained on the basis of augmented thyroidal rT₃ secretion. Study of rT₃-protein binding by equilibrium dialysis also failed to show any evidence of unusual rT₃-protein interaction (rT₃ free fraction was 2 — 3 times greater than in normal human serum). Among all the species examined so far (man, monkey, sheep, dog and rat), only in the dog are the circulating rT₃ levels significantly higher than T₃ suggesting that in this species the 5-deiodination, in marked contrast to the 5'-deiodination noted in several other species, is a major pathway normally involved in the initial monodeiodination of T₄.     

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.