Brian C. W. Hummel

5'-Iodothyronine deiodinase of rat liver: activity in microsomes prepared by various methods, solubilization by detergents and partial purification.

Iodothyronine deiodinase activities of rat liver microsomes prepared by (1) differential centrifugation, (2) column chromatography, (3) precipitation with Ca2+, (4) precipitation at low pH, or combinations of these were compared. Method 2 or 2 followed by 4 provided microsomes with specific activities 4.6- and 7.4-times higher than method 1, respectively. Both Triton X-100 at 0.1% (w/v) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) at 4-6 mM efficiently solubilized deiodinase and were not inhibitory at low concentrations. The Chaps-soluble enzyme could be moderately purified by fractionation with ammonium sulfate but more effectively with poly(ethylene glycol).

Improved α-glycerophosphate dehydrogenase assay system suitable for continuous recording

Abstract Improvements have been made in the Lee and Lardy assay for mitochondrial α-glycerophosphate dehydrogenase. Sonication of mitochondria in 1% Triton X-100 assures uniform dispersion of the particles and prevents precipitation of formazan, thus allowing continuous recording of the progress of the reaction.

Age-related changes in rat hepatic and renal thyroid hormone-sensitive enzymes — different responses to acute and chronic l-triiodothyronine stimulation

Using young (5-6 weeks) and adult (12-14 months) male Sprague-Dawley rats, the responses of hepatic and renal cytosolic malic enzymes (ME) and mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPDH), have been assessed following acute and chronic L-triiodothyronine (T3) administration. In control (untreated) animals a significant reduction of renal ME as well as hepatic and renal alpha-GPDH activity with increased age were observed but renal ME activity was not age-related. Following acute T3 stimulation (200 micrograms T3/100 g body wt single injection), the levels of hepatic ME and alpha-GPDH as well as renal alpha-GPDH but not renal ME remained lower in the adult than in the younger group. After chronic T3 stimulation (15 micrograms T3/100 g body wt for 7 days) or 200 micrograms T3/100 g body wt for 4 days), the enzyme levels remained significantly lower in older animals for hepatic ME but were no longer different for hepatic and renal alpha-GPDH, while renal ME, which was not altered with age, had values that were the same as in the younger group. These studies have demonstrated that age-related changes in hepatic and renal T3-sensitive enzymes could not be attributed solely to T3 occupancy of nuclear receptor binding sites, but may be influenced by other factors depending upon the specific tissues and subcellular T3-sensitive enzymes being assessed.

NADPH with Cytosol Stimulates Deiodination by Detergent-Solubilized Hepatic Microsomes: Evidence for NADPH-Dependent Cytosolic Non-Glutathione Reductase System

Based upon reconstitution experiments in which very low 5’-deiodinase (5’-DI) activity of isolated rat liver microsomes was restored to various degrees by the addition of cytosol, the existence of an endogenous cytosolicstimulating cofactor has been previously demonstrated (1). From similar reconstitution experiments using starved rats, it has been proposed that NADPH and/or GSH were cofactors for 5’-DI, the augmenting action of NADPH, being attributed to the generation of GSH through glutathione reductase (2). However, supporting evidence indicating that glutathione, as well as NADPH are essential endogenous cofactors in mediating 5’-DI stimulation has been controversial (3,4). We previously observed an important role of NADPH in stimulating microsomal 5’-DI in the presence of cytosol (5). Furthermore, using a microsome preparation, we have recently demonstrated a new non-glutathione NADPH-dependent cytosolic reductase system, which operates in the presence of intermediate (fraction B) and high molecular weight (MW) components (fraction A), without very low M.W. components including glutathione (GSH) (5). On the other hand, our laboratory has recently achieved solubilization of 5’-DI by detergents (6) and partial purification of the enzyme by DEAE-Sephacel column chromatography. Accordingly, it was the purpose of the present investigation to examine the effect of NADH, NADPH, and GSH on the stimulation of 5’-DI in a reconstitution assay system utilizing a detergent solubilized 5’-DI preparation with cytosol or fractionated cytosolic components (i.e., fraction A and B).

Generated Dihydrolipoamide and Cytosolic Components Stimulate Hepatic Microsomal Thyroxine 5’-Deiodination: Similar Effects of Cytosolic Components on Other Sulfhydryl Compounds

5′-deiodinase (5′-DI) is a thiol-dependent enzyme (1,2) and sulfhydryl compounds, such as dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and reduced glutathione (GSH, molecular weight [M.W.] 307 Da.) have been shown to function as activators by acting as direct reductants of the enzyme SH groups oxidized during deiodination. While several investigators have reported that isolated hepatic microsomes have very little 5′-DI activity in the absence of added thiols, reconstitution with cytosol has been reported to enhance 5′-DI activity, suggesting that cytosol contains one or more unidentified factors which are involved in 5′-deiodination (3). Recently, we have reported that cytosolic components of approximate M.W. 13,000 Da. (fraction B), which we have assigned an equivalent abbreviation of IMCC, might play a role as a direct reductant in an NADPH-dependent non-glutathione cytosolic reductase system (4). Hence, while sulfhydryl compounds may act directly with the 5′-DI, it is also possible that endogenous cytosolic factors may function as regulatory intermediaries between the enzyme and sulfydryl compounds, and that the overall influence of such compounds on 5′-DI may be mediated by such cytosolic cofactors.