Fumihide Isohashi

Immunochemical studies on malate dehydrogenase (decarboxylating) (NADP)

Abstract 1. 1. Malate dehydrogenase (decarboxylating) (NADP) ( l -malate:NADP oxidoreductase (decarboxylating), EC 1.1.1.40) was purified from the supernatant fraction of rat liver. 2. 2. Antibody against this purified enzyme was prepared. This antibody inhibited the activity of the enzyme and precipitated it. 3. 3. Malate dehydrogenase (decarboxylating) (NADP) in supernatant fractions from kidney, heart, brain and adipose tissue of rats reacted with the antibody in the same manner as that of the ra tliver enzyme in the inhibition tests. Supernantant malate dehydrogenases from all these tissues except adipose tissue formed single precipitin bands with the antibody on Ouchterlony double-diffusion analysis which fused with each other. 4. 4. The enzyme from the livers of different species differed immunologically from that of rat liver. 5. 5. Mitochondrial enzyme was immunologically quite distinct from supernatant enzyme. 6. 6. Increase and decrease in enzyme activities in the cytoplasm of liver under various dietary and hormonal conditions were accompanied by proportional changes in the quantity of immunologically reactive protein.

ARA24/Ran enhances the androgen-dependent NH2- and COOH-terminal interaction of the androgen receptor

The androgen receptor (AR) acts as an androgen-dependent transcription factor controlling the development of prostate tissue. Upon binding to androgen, AR undergoes a dynamic structural change leading to interaction between the NH(2)- and COOH-terminal regions of AR (N-C interaction). ARA24/Ran, which is a small GTPase, functions as an AR coactivator. Here, we report that ARA24/Ran enhances the N-C interaction of AR. The constitutively GTP- or GDP-bound form of ARA24/Ran repressed the AR N-C interaction. ARA24/Ran did not enhance the transcriptional activities of AR mutants that disrupt the N-C interaction. ARA24/Ran formed an endogenous protein complex with nuclear AR, but not cytoplasmic AR. Unlike SRC-1 with the positive activity for AR N-C interaction, ARA24/Ran did not enhance the transcriptional activity of the COOH-terminal domain-deleted AR mutant that is constitutively localized in the nucleus. These data demonstrate that ARA24/Ran increases AR transactivation by enhancing the AR N-C interaction in the nucleus.

Three forms of macromolecular translocation inhibitor of the nuclear binding of "activated" receptor-glucocorticoid complex and their interaction with ATP.

Three macromolecular translocation inhibitors of nuclear translocation or binding of “activated” receptor-[3H]triamcinolone acetonide in rat liver cytosol were eluted from a DEAE-cellulose column with a linear gradient of NaCl. The macromolecular material, eluted first from the column with about 0.1 M NaCl (Peak I), had the inhibitory activity, but in the presence of 5 mM ATP this fraction enhanced the nuclear binding. The inhibitory effect of the second peak (Peak II), eluted with about 0.2 M NaCl, was abolished by the addition of ATP. The inhibitor in Peak III, eluted with about 0.3 M NaCl, was not influenced by ATP. These results suggest that there are at least three inhibitors in rat liver cytosol, and that they are distinct entities, judging from their positions of elution on chromatography and the effects of ATP on their actions.

Macromolecular Translocation Inhibitor II (Zn2+-binding Protein, Parathymosin) Interacts with the Glucocorticoid Receptor and Enhances Transcription in Vivo

Macromolecular translocation inhibitor II (MTI-II), which was first identified as an in vitro inhibitor of binding between the highly purified glucocorticoid receptor (GR) and isolated nuclei, is an 11.5-kDa Zn2+-binding protein that is also known as ZnBP or parathymosin. MTI-II is a small nuclear acidic protein that is highly conserved in rats, cows, and humans and widely distributed in mammalian tissues, yet its physiological function is unknown. To elucidate its in vivo function in relation to GR, we transiently transfected mammalian cells with an expression plasmid encoding MTI-II. Unexpectedly, we found that the expression of MTI-II enhances the transcriptional activity of GR. The magnitude of the transcriptional enhancement induced by MTI-II is comparable with that induced by the steroid receptor coactivator SRC-1. In contrast, MTI-II had little effect on the transcriptional activity of estrogen receptor. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, GR coprecipitates with MTI-II, and, vice versa, MTI-II coprecipitates with GR. The expression of various deletion mutants of MTI-II revealed that the central acidic domain is essential for the enhancement of GR-dependent transcription. Microscopic analysis of MTI-II fused to green fluorescent protein and GR fused to red fluorescent protein in living HeLa cells showed that MTI-II colocalizes with GR in discrete subnuclear domains in a hormone-dependent manner. Coexpression of MTI-II with the coactivator SRC-1 or p300 further enhances GR-dependent transcription. Immunoprecipitation analysis showed that in the presence of glucocorticoid hormone, p300 and CREB-binding protein are coprecipitated with MTI-II. Furthermore, the knockdown of endogenous MTI-II by RNAi reduces the transcriptional activity of GR in cells. Moreover, expression of MTI-II enhances the glucocorticoid-dependent transcription of the endogenous glucocorticoid-inducible enzyme in cells. Taken together, these results indicate that MTI-II enhances GR-dependent transcription via a direct interaction with GR in vivo. Thus, MTI-II is a new member of the GR-coactivator complex.

Interaction of ATP with a macromolecular translocation inhibitor of the nuclear binding of “activated” receptor-glucocorticoid complex

Abstract Incubation of 1–5 mM ATP with nuclei and partially purified “activated” receptor-[3H]triamcinolone acetonide complex from rat liver cytosol had no significant effect on association of the activated complex with the nuclei. However, when the nuclear uptake was reduced by the macromolecular translocation inhibitor in the rat liver cytosol, addition of 5 mM ATP restored the uptake to the level without inhibitor. ADP and AMP as well as other nucleotides tested could not overcome the inhibitory effect of macromolecular inhibitor.

Effects of D- and L-thyroxine on the glucocorticoid binding capacity of adult rat liver.

Administration of either D- or L-thyroxine (T4) significantly increased the glucocorticoid binding capacity of cytosol of the livers of adrenalectomized adult rats. Administration of up to 0.5 mg/100 g body wt. of L-T4 was more effective than that of D-T4, but higher doses (0.8-3 mg/100 g body wt.) of D-T4 increased the binding capacity markedly to more than that with L-T4. T4- administration did not alter the apparent dissociation constant of glucocorticoid binding proteins for glucocorticoid binding, or their behavior on DEAE-cellulose chromatography either before or after thermal activation (23 degrees C for 40 min). Thus the increased binding capacity seemed to be due to increase in the level of glucocorticoid receptor in rat liver.

Regulation of nuclear uptake of “Activated” receptor-glucocorticoid complex by pyrophosphate

Nuclear uptake of partially purified “activated” receptor-[ 3 H]triamcinolone acetonide complex from rat liver cytosol in vitro was significantly increased by incubation with pyrophosphate, reaching a maximum with 2–7 mM pyrophosphate, but with higher concentrations of 9–10 mM pyrophosphate it decreased gradually to below the control level. When nuclear uptake of “activated” complex was inhibited by macromolecular translocation inhibitor, ATP could overcome the inhibitory effect of macromolecular inhibitor, while 1–5 mM pyrophosphate could not enhanced nuclear uptake. Addition of 1–5 mM phosphate did not affect nuclear uptake or the action of the macromolecular translocation inhibitor.

Simple and unique purification by size-exclusion chromatography for an oligomeric enzyme, rat liver cytosolic acetyl-coenzyme A hydrolase

An overview of the purification of an oligomeric enzyme, an extramitochondrial acetyl-coenzyme A hydrolase from rat liver, is presented. The enzyme has been purified to homogeneity using two successive size-exclusion chromatography runs, first for the monomeric and second for the oligomeric form of the enzyme. The sequential gel-filtration steps efficiently removed the contaminants of any molecular size, first of different size from that of the monomeric form of the enzyme (K(av)=0.47 on Superdex 200) and second of different size from that of the oligomeric form (K(av)=0.33), allowing us to purify the enzyme in high purity. This strategy provides an excellent model for purifying many other oligomeric proteins including key enzymes or allosteric enzymes regulating metabolism.

New purification method for glucocorticoid receptors

In this report, we describe a new purification method for activated recombinant glucocorticoid receptor (GR) utilizing a cation-exchanger (Mono S) at pH8.4. This method is based upon a new finding that activated GR binds to both Mono Q and Mono S columns at the same pH. This method enables us to purify recombinant GR within 3 h. The purified GR represents more than 97% of the eluted proteins. Purified recombinant GR is able to bind specifically to a DNA fragment containing the glucocorticoid response element. Recombinant GR has no tag sequence that can be utilized for purification. Thus, this separation method is also applicable to purification of native GR.

Novel and simple two-step purification of a full-length rat glucocorticoid-receptor expressed in a baculovirus system

We purified the activated recombinant glucicorticoid receptor (GR) overexpressed in insect cells by sequential chromatographies using Mono Q and Mono S columns. This procedure was based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH (pH 8.4). The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. The purified GR was able to bind specifically to a DNA fragment containing the glucocorticoid response element. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.