Comparison of the physicochemical properties of type I and type II iodothyronine 5'-deiodinase

The 5'-deiodination of thyroxine is catalyzed by two enzymes which differ in their tissue distribution, substrate specificities, sensitivity to the inhibitor, propylthiouracil, and response to thyroid status. By using the affinity label, N-bromoacetyl-L-thyroxine, both isoenzymes have been found to have substrate binding subunits of approximately 27 kDa. In this study, we compared the substrate binding subunits and hydrodynamic properties of the type I and the type II isozymes using the affinity label, N-bromoacetyl-L-thyroxine, to identify the enzymes. High resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the substrate binding subunit of the type I enzyme had an Mr of 27,000, while that of the type II enzyme had a slightly higher Mr of 29,000. This difference was not accounted for by glycosylation. Partial staphylococcal V8-protease digests of the substrate binding subunit of the type I enzyme yielded fragments of 14.6, 13.7, and 7.0 kDa, while V8-protease digests of the substrate binding subunit for the type II enzyme produced fragments of 28.0, 25.1, 19.0, 9.5, 7.2, and 5.8 kDa. Unique cyanogen bromide fragmentation patterns were also observed for the two substrate binding subunits. Sedimentation coefficients of the detergent-soluble type I and type II holoenzymes were 3.67 and 5.22 S, respectively, as determined by sucrose density centrifugation. The type I enzyme behaved as a globular protein, whereas the type II enzyme showed sedimentation properties typical of asymmetric integral membrane proteins. The Stokes radii were 3.78 and 4.97 nm, respectively. From these data, the calculated Mr for detergent-solubilized type I 5'-iodothyronine deiodinase was 55,400 and for the type II enzyme was 198,700. These data indicate that the two isozymes of iodothyronine 5'-deiodinase are multimeric, differ in holoenzyme size and subunit composition, and that their substrate binding subunits are distinct.     

Evidence that pimozide is not a partial agonist of dopamine receptors.

The dopamine receptor antagonist pimozide, at concentrations up to 10 nM, competitively antagonized the inhibitory action of a pomorphine on prolactin (PRL) secretion by cultured rat pituitary cells. At higher concentrations pimozide as well as the analogues clopimozide and penfluridol suppressed PRL secretion. The latter effect could not be reversed by dopamine antagonists devoid of intrinsic effects on PRL release. Suppression of PRL release was also observed with compounds which were devoid of dopamine receptor agonistic or antagonistic properties such as R 6694 and R 5052, structurally related to pimozide, and also with loperamide. The inhibitory action of pimozide on PRL release resembled that of the calcium antagonist flunarizine. Concentration effect curves showed parallel slopes and the effect of both compounds could be reversed by increasing the concentration of calcium ions (Ca²⁺). Both flunarizine and pimozide were also capable of inhibiting releasing factor-stimulated luteinizing hormone secretion, an effect not shared by apomorphine. Pimozide and the various structurally related compounds used, also antagonized Ca²⁺-induced smooth muscle contractions of the isolated caudal artery of the rat.The present findings indicate that pimozide is a competitive antagonist without partial agonistic activity on apomorphine-sensitive dopamine receptors in the pituitary and that its inhibitory effect on PRL release as well as on vascular smooth muscle contractions is due to interference with a Ca²⁺-dependent mechanism of the stimulus-effect coupling process.     

Identification of type I iodothyronine 5'-deiodinase as a selenoenzyme

A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'-deiodinase subunit, affinity-labeled with N-bromoacetyl-[125I]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme.     

Evidence that rat pineal thyroxine 5'-deiodinase is primarily stimulated by beta- and not alpha-adrenergic agonists and that its adrenergic-stimulated and spontaneous rhythmic nocturnal rise require RNA and protein synthesis

Pineal thyroxine 5'-deiodinase (5'-D) activity rose greater than 10-fold above the basal level 2-3 hr after 1 mg/kg isoproterenol and returned to near the basal level by 6 hr. The same dose of norepinephrine or phenylephrine was without effect, but phenylephrine modestly potentiated isoproterenol-stimulated 5'-D activity. Either actinomycin D or cycloheximide treatment markedly decreased diurnal isoproterenol stimulation and the spontaneous rhythmic nocturnal rise of pineal 5'-D. The data indicate that pineal 5'-D activity is very similar to pineal serotonin N-acetyl transferase in being primarily stimulated by beta-adrenergic agonists and requiring new RNA and protein synthesis for its activation.     

Evidence that mutacin II production is not mediated by a 5.6-kb plasmid in Streptococcus mutans

Here we present evidence that the cryptic 5.6-kb plasmid found in certain strains of Streptococcus mutans is not involved in mutacin production. This evidence comes from demonstrating similarities between a plasmid-less strain T8 and a group II plasmid strain UA96. Both produce what appears to be an identical mutacin based on spectrum of activity and physiological properties. Also, T8 and UA96 are members of the same immunity group (group II). Genotypically, both strains appear similar except for plasmid content based on DNA fingerprinting profiles. T8 and UA96 exhibit identical hybridization patterns following transformation of T8 with a mutacin-negative (bac-1::Tn916) sequence from a Tn916-insertionally inactivated mutant of UA96. This transformation also resulted in the mutacin-negative phenotype in T8 transformants, showing recombination between a mutacin-associated gene in UA96 and its apparent homologous sequence in T8. Moreover, when a plasmid containing a putative repeat element from UA96 (pPC264) was used as a probe, it hybridized to the same five EcoRI fragments in both T8 and UA96. Collectively, these data, coupled with data from other sources, indicate that the plasmid resident in mutacin II strains is not involved in mutacin production.     

Mechanisms of chromosome banding. II. Evidence that histones are not involved

C-and G-banding in mouse metaphase chromosomes is unaffected by exposure of the chromosomes to 0.2 N HCl for 4 h. Electrophoretic studies indicate that this is adequate to completely remove the histones from fixed and dried chromatin thus indicating that histones are not involved in C- or G-banding.     

Evidence that type II insulin-like growth factor receptor is coupled to calcium gating system

In competent Balb/c 3T3 cells primed with epidermal growth factor (primed competent cells), insulin-like growth factor-II (IGF-II) stimulated calcium influx in a concentration dependent manner with the ED50 of 450 pM. When receptor-bound [125I]IGF-II was cross-linked by use of disuccinimidyl suberate, a 240 K-Da protein was radiolabeled. Excess amount of unlabeled IGF-II inhibited the affinity-labeling of the 240 K-Da protein. To further examine whether IGF-II stimulates calcium influx by acting on the type II IGF receptor, we employed polyclonal antibody raised against rat type II IGF receptor, R-II-PABl. This antibody immunoprecipitated the type II IGF receptor and inhibited IGF-II binding in Balb/c 3T3 cell membrane without affecting IGF-I binding. In primed competent cells, R-II-PABl elicited an agonistic action in stimulating [3H]thymidine incorporation. Under the same condition, R-II-PABl elicited a marked stimulation of calcium influx. These results suggest that, in Balb/c 3T3 cells, 1) relatively low concentrations of IGF-II act mainly on the type II IGF receptor; 2) the type II IGF receptor is coupled to a calcium gating system; and 3) binding of a ligand to the type II IGF receptor leads to the stimulation of DNA synthesis.     

Evidence that NiNi acetyl-CoA synthase is active and that the CuNi enzyme is not

The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) plays a central role in the Wood-Ljungdahl pathway of autotrophic CO(2) fixation. One structure of the Moorella thermoacetica enzyme revealed that the active site of ACS (the A-cluster) consists of a [4Fe-4S] cluster bridged to a binuclear CuNi center with Cu at the proximal metal site (M(p)) and Ni at the distal metal site (M(d)). In another structure of the same enzyme, Ni or Zn was present at M(p). On the basis of a positive correlation between ACS activity and Cu content, we had proposed that the Cu-containing enzyme is active [Seravalli, J., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3689-3694]. Here we have reexamined this proposal. Enzyme preparations with a wider range of Ni (1.6-2.8) and Cu (0.2-1.1) stoichiometries per dimer were studied to reexamine the correlation, if any, between the Ni and Cu content and ACS activity. In addition, the effects of o-phenanthroline (which removes Ni but not Cu) and neocuproine (which removes Cu but not Ni) on ACS activity were determined. EXAFS results indicate that these chelators selectively remove M(p). Multifrequency EPR spectra (3-130 GHz) of the paramagnetic NiFeC state of the A-cluster were examined to investigate the electronic state of this proposed intermediate in the ACS reaction mechanism. The combined results strongly indicate that the CuNi enzyme is inactive, that the NiNi enzyme is active, and that the NiNi enzyme is responsible for the NiFeC EPR signal. The results also support an electronic structure of the NiFeC-eliciting species as a [4Fe-4S](2+) (net S = 0) cluster bridged to a Ni(1+) (S = (1)/(2)) at M(p) that is bridged to planar four-coordinate Ni(2+) (S = 0) at M(d), with the spin predominantly on the Ni(1+). Furthermore, these studies suggest that M(p) is inserted during cell growth. The apparent vulnerability of the proximal metal site in the A-cluster to substitution with different metals appears to underlie the heterogeneity observed in samples that has confounded studies of CODH/ACS for many years. On the basis of this principle, a protocol to generate nearly homogeneous preparations of the active NiNi form of ACS was achieved with NiFeC signals of approximately 0.8 spin/mol.     

The actin cytoskeleton mediates the hormonally regulated translocation of type II iodothyronine 5'-deiodinase in astrocytes

Thyroid hormone, specifically thyroxine, alters cytoskeletal organization in astrocytes by modulating actin polymerization and, in turn, regulates the turnover of the short-lived membrane protein, type II iodothyronine 5'-deiodinase. In the absence of thyroxine, approximately 35% of the total cellular actin is depolymerized, and greater than 90% of the deiodinase is found in the plasma membrane and not associated with the cytoskeleton. Addition of thyroxine promotes actin polymerization and decreases the depolymerized actin to approximately 10% of the total actin pool, induces binding of the deiodinase to F-actin, and promotes rapid internalization of the enzyme. These data provide direct evidence that the actin cytoskeleton participates in the inactivation pathway of the deiodinase by translocating this short-lived plasma membrane protein to an internal membrane pool.     

Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant.

Previous analyses have demonstrated that adenovirus DNA is packaged into virions in vivo in a polar, left-to-right fashion. The packaging of viral DNA is dependent on cis-acting elements at the left end of the genome. In this report, we describe a genetic analysis of the sequences that are required for efficient packaging of adenovirus type 5 (Ad5) DNA. Our results demonstrate that the Ad5 packaging domain (nucleotides 194 to 358) is composed of at least five distinct elements that are functionally redundant. An AT-rich repeated sequence motif, the A repeat, is located in four of five of these regions; the fifth region is also AT rich. The efficiency of viral packaging depends on the number of individual A repeats that are present in the viral genome. The deletion of the entire packaging domain resulted in the loss of virus viability. A virus that contains a multimerized oligonucleotide corresponding to A repeat II in place of the packaging domain could package viral DNA, although with reduced efficiency compared with that of the wild-type virus. Our results also suggest that the spacing of specific sequences at the left end of the Ad5 genome are important for enhancer region function in vivo.     

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

It has been suggested that the infectious agents of scrapie and Creutzfeldt-Jakob disease (CJD) are 'prions' constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re-evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase-K-resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one-half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.     

Beta- and alpha-adrenergic receptors are involved in regulating type II thyroxine 5'-deiodinase activity in the rat Harderian gland.

The role of alpha- and beta-adrenergic receptors in regulation of rat Harderian gland type II thyroxine 5'-deiodinase (5'-D) activity was investigated. Our results show that isoproterenol, a beta-adrenergic agonist, and phenylephrine, an alpha-adrenergic agonist, elicited increases in Harderian gland 5'-D activity. The activation was dependent on the time and the dose of the drug. Other adrenergic agonists, i.e., norepinephrine, methoxamine or terbutaline, also clearly increased the enzyme activity. Moreover, administration of propranolol, a beta-adrenergic blocker, or prazosin, an alpha-adrenergic blocker, completely prevented the activation of the enzyme induced by norepinephrine. Results show a clear regulation by adrenergic mechanisms of 5'-D activity in the rat Harderian gland, where alpha- and beta-adrenergic receptors appear to be involved.     

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol

Herein we show, for the first time, a very marked increase in thyroxine 5'-deiodinase (5'-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5'-D than was isoproterenol, suggesting that, in addition to beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5'-D activity. The nocturnal increase in pineal 5'-D activity was completely abolished by propranolol, a beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.