The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.     

Movement of water in conjunction with plant movement visualized by NMR imaging

The water distribution in the pulvinus of Mimosa can be visualized by an NMR imaging technique. After stimulation of a Mimosa plant, water in the lower half of the main pulvinus disappeared, the water previously contained in this area seeming to be transferred to the upper half of the main pulvinus. Movement of the water in conjunction with Mimosa movement was visualized sequentially by a non-invasive NMR imaging procedure.     

The effect of inosine on the post ischemic heart as bio-energy recovering factor in 31P-MRS

Perfused guinea-pig hearts, which were analyzed by 31P-MRS, were subjected to 30 and 60 minute ischemia and reperfused using two perfusates, one containing 200 microM inosine, and the other without inosine. After 4 hour reperfusion with inosine, ATP levels increased to 95.5% of preischemic value (30 minute ischemia) and 76.2% (60 minute ischemia). However, after 4 hour reperfusion without inosine, ATP levels increased only to 72.2% (30 minute ischemia) and to 48.2% (60 minute ischemia). In 60 minute ischemic hearts reperfused with inosine, left ventricular maximal positive dp/dt (LV dp/dt) was improved significantly to 82.4% after 6 hour reperfusion in contrast to hearts reperfused without inosine (43.1%). Administration of inosine was very useful for increasing myocardial gross energy product and improving cardiac performance.     

The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X

A major isozyme of rat heart glutathione transferase was purified to homogeneity by Sephadex G-200 gel filtration, ammonium sulfate precipitation, CM-cellulose chromatography and affinity chromatography on S-hexylglutathione-linked Sepharose 6B. The purified isozyme was a dimer with an apparent relative molecular mass of 50 000 composed of two Yb-size subunits (Mr = 26 500). The isozyme is immunologically related to rat liver glutathione transferase X and 3-3, especially closely to transferase X, and no immunological cross-reactivity with subunits 1 and 2 of hepatic glutathione transferases was observed. The isoelectric point (pI = 6.9) of the isozyme was identical with and the substrate specificity was very similar to transferase X. Thus, the cardiac near-neutral isozyme is considered to be identical to glutathione transferase X recognized in rat liver. The amount of this near-neutral isozyme estimated to be present in heart tissue is 70 micrograms/g. The isozyme has relatively high activities towards alpha, beta-unsaturated carbonyl compounds such as trans-4-phenyl-3-buten-2-one and trans-4-hydroxynon-2-enal. The latter is a cytotoxic product resulting from lipid peroxidation of polyunsaturated fatty acids, and the cardiac isozyme may play a physiologically significant role with glutathione conjugation of this compound. In addition to the near-neutral isozyme, acidic forms with isoelectric points of 4.9, 5.2 and 5.5 were partially purified; some of them are considered to consist of subunits immunologically related to transferase X.     

Structure of the trypsin-binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.     

Atrial natriuretic factor increases cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubule cells in culture

The present study was undertaken to determine whether human atrial natriuretic factor (hANF) produces guanosine-3', 5'-monophosphate (cGMP) and alters arginine vasopressin (AVP)- and forskolin (F)- induced adenosine-3', 5'-monophosphate (cAMP) production in the cultured rat renal papillary collecting tubule cells. hANF increased cellular cGMP levels in a dose dependent manner. AVP and F, however, did not affect cGMP production. hANF significantly inhibited AVP- and F-stimulated cAMP levels, but hANF by itself did not affect cellular cAMP production. Since F activates adenylate cyclase at a step of catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic units, the present results indicate that hANF may directly inhibit the AVP- and F-stimulated adenylate cyclase in renal papillary collecting tubules.     

Liquid chromatographic determination of free and conjugated 3-methoxy-4-hydroxyphenylethylene glycol with wide-ranging substances related to monoamine metabolism

A simple and sensitive procedure was developed for the simultaneous determination of substances metabolically related to monoamine transmitters including 3-methoxy-4-hydroxy-phenylethylene glycol (MOPEG) in dissected brain regions of rats using high-performance liquid chromatography combined with electrochemical detection. The tissue sample was homogenized in HCl solution. The homogenate was divided into two portions, of which one was used for the assay of MOPEG after enzymatic hydrolysis with sulfatase. A butanol extraction process was performed on the remaining portion to obtain the sample of monoamine transmitters, precursor amino acids, and acidic metabolites. The monoamines and precursor amino acids were finally recovered in HCl solution, while the acidic metabolites shifted into the alkaline buffer from the organic layer. The basic and neutral substances were separated with a 0.1 M sodium citrate/citric acid buffer system (pH 4.0) containing 1% tetrahydrofuran, and the acidic ones with 0.075 M sodium citrate/citric acid buffer (pH 3.5) containing 1% tetrahydrofuran, 10% methanol, and 12% acetic acid. The steady-state concentrations of three monoamine transmitters (noradrenaline, dopamine, and 5-hydroxytryptamine) were determined together with their precursors and metabolites. Changes in the concentrations of these substances were examined for various drugs, of which the effects had been previously confirmed. The changes reflected putative drug effects and demonstrated that the procedure was applicable to the regional determination of monoamines and their metabolically related substances.     

Activated c-raf gene in a rat hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.