Effects of the phosphodiesterase-IV inhibitor EMD 95832/3 on tumour growth and cachexia in rats bearing the Yoshida AH-130 ascites hepatoma.

Administration of the phosphodiesterase-IV inhibitor EMD 95832/3 (Merck KGaA, Darmstadt, Germany) to rats bearing the ascites hepatoma Yoshida AH-130, a highly cachectic tumour, could not prevent either the anorexia nor the massive weight loss (affecting both adipose and skeletal muscle tissues) present in the tumour-bearing animals. This compound did not have any effects on the fractional rates of protein turnover in skeletal muscle, and did not affect circulating triacylglycerols or lipoprotein lipase activity in adipose tissue. Although the administration of EMD 95832/3 did not influence tumour growth either, it did increase the number of tumour cells undergoing apoptosis. It is concluded that the drug is unable to reverse the cachectic state in this particular experimental tumour model.

Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin-proteasome-dependent proteolysis in cancer and septic rats.

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-alpha (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14 kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19 S regulatory complex of the 26 S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub-proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

[Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use]. / Citotoxicidad de la beta-lapachona: una o-naftoquinona con posibles usos terapéuticos.

beta-lapachone (beta-lap) is a lipophilic o-naphthoquinone isolated from the bark of the lapacho tree. Initial observations proved its capability for inhibiting growth of Yoshida tumor and Walker 256 carcinosarcoma. beta-Lap redox-cycling in the presence of reductants and oxygen yields "reactive oxygen species" (ROS: O2-, OH and H2O2) which cytotoxicity led to assume its role in beta-lap activity in cells. beta-Lap inhibited DNA synthesis in Trypanosoma cruzi as well as topoisomerases I and II, poly(ADP-ribose) polymerase (PARP) in different cells. These enzymes are essential for maintaining DNA structure. beta-Lap inhibited growth of a large variety of tumor cells including epidermoid laringeal cancer, prostate, colon, ovary and breast cancer and also different types of leukemia cells. Advances in knowledge of apoptosis ("programmed cell death") and necrosis provided useful information for understanding the mechanism of beta-lap cytotoxicity. Thiol-dependent proteases (Calpaine), kinases (e.g. c-JUN NH2-terminal kinase), caspases and nucleases are involved in beta-lap cytotoxicity. These enzymes activity, as well as ROS production by beta-lap redox-cycling, would be essential for beta-lap cytotoxicity. Diaphorase and NAD(P)H-quinone reductase, which catalyse beta-lap redox-cycling and ROS production, seem to play an essential role in beta-lap activity. On these grounds, clinical applications of beta-lap have been suggested.

Citotoxicidad de la ß-Lapachona: una O-Naftoquinona con posibles usos terapéuticos / Citotoxicity of ß-Lapachone: an O-naphthoquinone with possible therapeutic uses

La ß-lapachona (ß-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida, del carcinosarcoma de Walker 256 y del Trypanosoma cruzi. La ß-lap genera productos reactivos del oxígeno (EROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. ß-lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de las topoisomerasas I y II de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la reparación y mantenimiento de la estructura del ADN. Se investigó la citotoxicidad de ß-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y diferentes formas de leucemia aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de ß-lap y su relación con los procesos de apoptosis y necrosis. Entre esos mecanismos se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN), caspasas y nucleasas, que finalmente degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la ß-lap es su reducción enzimática, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de EROS. La acción de ß-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres generados por la ß-lap. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer. (AU)

Citotoxidad de la beta lapachona: una o-naftoquinona con posibles usos terapeuticos / Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use

La Beta-lapachona (Beta-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida y del carcinosarcoma de Walker 256. La Beta-lap genera productos reactivos del oxígeno (ROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. Beta-Lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de la topoisomerasas I y II y de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la conservación del ADN. Se investigó la citotoxicidad de Beta-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y leucemia, aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de Beta-lap y su relación con los procesos de apoptosis y de necrosis. Se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN NH2 -quinasa terminal), caspasas y nucleasas, enzimas que degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la Beta-lap es su reducción, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de ROS. La acción de Beta-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer. (Au)

Citotoxidad de la beta lapachona: una o-naftoquinona con posibles usos terapeuticos / Cytotoxicity of beta-lapachone, an naphthoquinone with possible therapeutic use

La Beta-lapachona (Beta-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida y del carcinosarcoma de Walker 256. La Beta-lap genera productos reactivos del oxígeno (ROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. Beta-Lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de la topoisomerasas I y II y de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la conservación del ADN. Se investigó la citotoxicidad de Beta-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y leucemia, aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de Beta-lap y su relación con los procesos de apoptosis y de necrosis. Se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN NH2 -quinasa terminal), caspasas y nucleasas, enzimas que degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la Beta-lap es su reducción, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de ROS. La acción de Beta-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer.

Stimulation of brown adipose tissue activity in tumor-bearing rats.

Despite anorexia, cancer development is frequently accompanied by an increase of energy expenditure. Considering the pivotal role played by brown adipose tissue (BAT) in the energy metabolism of small mammals, we investigated the functional and compositional modification in BAT of anorexic tumor-bearing (Yoshida sarcoma) and pair-fed control rats. BAT thermogenic activity (assessed by maximal mitochondrial GDP binding) was 1.8-fold greater in tumor-bearing rats than in controls, while the thermogenic capacity (assessed by measurement of uncoupling protein) was unchanged. This suggests that tumor bearing had induced an unmasking of uncoupling protein sites. BAT hypertrophy and hyperplasia, characteristic of full-fledged BAT activation, did not occur. The mitochondrial oxidative capacity of BAT (assessed by cytochrome c oxidase activity) was 1.6-fold lower in tumor-bearing than in control rats. The main compositional modification observed in BAT of tumor-bearing rats was an increase in the saturation of cardiolipin fatty acids. These results suggest that the BAT stimulation induced by tumor bearing after 10 days is almost exclusively functional and that the tissue development is limited, probably by anorexia. However, a suppressive effect of anorexia inhibition by tumor bearing cannot be excluded.

Effects of glutamate dehydrogenase, choline oxidase, and glucose-6-phosphatase on 67Ga accumulation in lysosome.

AIM: To clarify the effects of the activities of hepatic enzymes in liver, hepatoma, and malignant tumor on 67Ga accumulation in lysosome. METHODS: 67Ga-citrate solution was prepared from carrier-free 67Ga-citrate solution 0.08 mol.L-1 and sodium citrate solution 0.08 mol.L-1, and was injected iv 0.4 ml to the rats. Subcellular fractions of the liver were measured for radioactivity of 67Ga by a well-type scintillation counter (Aloka JDC-701). Glutamate dehydrogenase, choline oxidase, and G-6-P activities were calculated as described by Shimizu H, Ikuta S, and Baginski E, respectivily. RESULTS: 67Ga radioactivity in normal liver lysosome (55%) was significantly higher than those in hepatoma AH 109A (32%) and Yoshida sarcoma (18%). Glutamate dehydrogenase activities were 1830 +/- s 320 U.L-1 in normal liver while 23 +/- s 6 U.L-1 in hepatoma AH 109A, and 7 +/- s 2 U.L-1 in Yoshida sarcoma. Choline oxidase activities were 46 +/- s 10 U.L-1 for normal, 25.0 +/- s 0.4 U.L-1 for hepatoma AH 109A, and 2.0 +/- s 0.4 U.L-1 for Yoshida sarcoma. G-6-P activities were 2550 +/- s 180 U.L-1 in normal, 84 +/- s 14 U.L-1 in hepatoma AH 109A, and 78 +/- s 13 U.L-1 in Yoshida sarcoma. CONCLUSION: Lysosome of normal rat liver in which hepatic enzymes work actively played a major role in the tissue concentration of 67Ga, but the role diminishes with the neoplastic transformation into hepatoma. The lysosome of Yoshida sarcoma does not play any role in 67Ga accumulation because it does not possess any features of liver.

Field bean protease inhibitor preparations, unlike methotrexate, can completely suppress Yoshida sarcoma tumor in rats.

Protease inhibitor preparations (PIP) with antitryptic and antichymotryptic activities, isolated from field bean legume as well as doxorubicin and cyclophosphamide could effectively suppress the growth of Yoshida sarcoma ascites tumor cells transplanted in adult rats and prevent their death. As against this, methotrexate and heat-inactivated PIP were ineffective in such rats at varied doses of treatment tried. The percent survival of animals appeared to be related to the purity, treatment mode and the dose of PIP used. Zymographic analysis of the trypsin activated sarcoma cell homogenate revealed the presence of six protease bands in the molecular weight range of 51kD to 206kD. Prolonged interactions of such zymograms with protease inhibitors such as 20mM EDTA or 5mM diisopropyl flurophosphate (DIFP) or 400 micrograms/ml of PIP in reaction buffer indicated that these are not metalloproteases but serine proteases whose activities are inhibited by PIP and DIFP. Since proteases are involved in cell growth regulation and cell transformation, we hypothesize a positive relationship between the field bean protease inhibitor's blocking action on tumor cell proteases and its tumor suppressing activity.

[Innate resistance to thymidylate synthase inhibition after 5-fluorouracil treatment--a rationale of combined use of cisplatin and its optimal administration dose].

We examined the changes of the number of FdU MP binding sites of thymidylate thynthase (TS-BS) in Yoshida sarcoma after administration of 5-FU to the tumor bearing rats. We also investigated the optimal dose of CDDP for the increase of intracellular folate level. In the group received consecutive 7-days administration of UFT (U-7 group), total TS-BS was significantly increased compared with non-treatment group and the group received only UFT (U-1 group). For free TS-BS, however, there was no difference despite of UFT administration. Thymidylate synthase inhibition rate (TSIR) was, therefore, significantly high in U-7 group compared with U-1 group. It seemed necessary to take some counter measure for the induction of TS in the tumor tissue when 5-FU chemotherapy was performed. The optimal dose of CDDP as a modulator of 5-FU was 1 mg/kg in rat when it was estimated from the changes of intracellular folate levels after administration, which was less than the dose to reveal its own anticancer effect.

Characterization of urokinase-type plasminogen activator of rat decidual tissue.

Urokinase-type plasminogen activator (uPA) from artificially induced decidual tissue of rat has been purified to homogeneity employing chromatographic techniques and the final preparation has a specific activity of 12,084 I.U./mg. The purified preparation resolves into a single band following SDS-PAGE and has an apparent molecular weight of 45 kDa. HPLC of the purified fraction also yields a single peak at 45 kDa. Decidual uPA is immunogenic in rabbit and a monospecific antiserum raised against it does not cross react with human melanoma tPA or rat Yoshida sarcoma tPA but elicits a precipitin reaction with human uPA and extracts of rat placenta and kidney. The enzyme has a pH optimum of 7.5, a kM of 1.0 microM, is heat stable upto ten minutes at 42 degrees C and inhibited by anti-uPA IgG.

Selective increases in isoform PP1 alpha of type-1 protein phosphatase in ascites hepatoma cells.

The amounts of four isoforms of the catalytic subunit of type-1 protein phosphatases, PP1 alpha, PP1 gamma 1, PP1 gamma 2, and PP1 delta, have been determined in extracts of rat ascites hepatomas, AH131A, AH13, AH13NMOR, AH143A, and Yoshida sarcoma, and compared to those of rat liver by Western blot analysis. The amount of PP1 alpha was increased over three times in all five hepatomas. The amount of PP1 gamma 1 was increased over two times in AH13, AH13NMOR, and AH143A. The amount of PP1 delta was selectively increased about 4 times in AH131A and AH143A. The PP1 gamma 2 protein was undetectable in both liver and hepatomas. There was good parallelism between the general increase in only PP1 alpha protein in the hepatomas and the previous data demonstrating the general increase in PP1 alpha mRNA in numerous ascites hepatomas. These results suggest that PP1 alpha plays important roles in the expression of malignant phenotype, that its amount is under strict control at the transcription level, and that PP1 gamma 1 and PP1 delta play different roles in the expression of some phenotype(s) of the ascites hepatomas.

3-Cyano-2,6-dihydroxypyridine (CNDP), a new potent inhibitor of dihydrouracil dehydrogenase.

3-Cyano-2,6-dihydroxypyridine (CNDP) was identified as a potent inhibitor (IC50 value, 4.4 nM) of dihydrouracil dehydrogenase (DHUDase) [EC 1.3.1.2], a rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. The inhibitory activity of CNDP was about 2,000 times that of uracil under our assay conditions. Kinetic analyses with partially purified enzyme from rat liver revealed that the mechanism of inhibition of DHUDase by CNDP was of mixed type with an inhibition constant (Ki) of 1.51 nM. CNDP had less effect on 5-FU phosphorylation than on 5-FU degradation. The inhibitory effect of CNDP on ribosylation of 5-FU was 600 to 1,000 times less than that on DHUDase. Moreover, CNDP did not inhibit uridine kinase, thymidine kinase, or pyrimidine phosphoribosyltransferase. Coadministration of CNDP with 1-ethoxymethyl-5-fluorouracil (EM-FU) to rats with Yoshida sarcoma elevated the level of 5-FU in both the blood and the tumor and enhanced the antitumor effect of EM-FU. These findings indicated that CNDP would be a useful chemical modulator in chemotherapy with 5-FU or its prodrugs.

Regulation of transglutaminase activity by GTP in digitonin permeabilized Yoshida tumor cells.

Yoshida tumor cells contain consistent amounts of type 2 transglutaminase, along with a membrane bound form of the enzyme. Digitonin permeabilized cells retain a large proportion of type 2 TGase and of substrate proteins which are labelled by radioactive putrescine in the presence of calcium. GTP inhibits protein labelling at low calcium concentration by inhibiting type 2 TGase without affecting membrane-bound TGase. These results support the notion that inhibition of type 2 TGase by GTP is physiologically relevant.

Isolation and purification of plasminogen activator from Yoshida ascites Sarcoma of rats.

The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33% saturation followed by affinity chromatography on p-aminobenzamidine-Sepharose 4B. The specific activity of the purified activator was 10,260 IU/mg expressed in terms of International units of urokinase, the known activator of plasminogen. The activator was homogeneous by polyacrylamide slab gel electrophoresis with an apparent molecular weight 75 kDa by gel filtration on Sephadex G-100. Analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, revealed the presence of two subunits of about 48 and 29 kDa. The activator displayed binding preference to fibrin and was immunologically distinguishable from urokinase, indicating that it could be of non-urokinase origin. The preparation further revealed similarity to standard tissue plasminogen activator with respect to fibrin binding and immunological cross reactivity.

Cartilage-degrading neutral proteinase secreted by Yoshida sarcoma cells. Purification and properties.

The Yoshida sarcoma, a malignant rat tumor, has been reported by Machinami ( Machinami , R. (1972) Acta Pathol. JPN 22, 19-39) to destroy cartilage matrix in vivo. We have characterized an enzyme secreted by Yoshida sarcoma cells in culture which degrades cartilage proteoglycan in solution and also in situ in organ culture ( Mikuni - Takagaki , Y., and Gross, J. (1981) in Proceedings of the 6th International Symposium on Glycoconjugates ( Yamakawa , T., Osawa , T., Handa , S., eds) pp. 491-492, Japan Scientific Societies Press, Tokyo) ( Mikuni - Takagaki , Y., and Gross, J. (1982) in The Extracellular Matrix ( Hawkes , S., and Wang, J.L., eds) pp. 379-385, Academic Press, New York). In this report we characterized the isolated enzyme with the help of a new assay system for measurement of proteoglycan core protein degradation, which utilizes aminopropyl glass beads derivatized with hyaluronic acid. This enzyme, with a neutral pH optimum and apparent molecular weight of about 30,000, is secreted into culture medium in an active form. It is resistant to cartilage-derived inhibitors and to alpha 2-macroglobulin as well as to synthetic and natural inhibitors of serine-, thiol- and carboxylproteinases . It is inhibited by a chelating agent, 1,10-phenanthroline and thiol compounds at relatively high concentrations, and therefore is probably a metalloproteinase. The enzyme degrades type V collagen, types I and II denatured collagen (gelatin), and casein in addition to cartilage proteoglycan, but not bovine serum albumin, myoglobin, fibrinogen, elastin or native collagen types I, II, III, and IV. These findings suggest that the Yoshida sarcoma may degrade cartilage matrix in vivo by means of a secreted, active, inhibitor-resistant enzyme.

[Regulation of enzyme activities involved in pyrimidine synthesis and its application to cancer chemotherapy].

We examined the enhancing effect of antitumor activity by co-administration of 5-FU or its masked form (FT-207) with bases or nucleosides of purine and pyrimidine. It was found that the antitumor activity of 5-FU or FT-207 on sarcoma-180 and AH-130 tumor was oral administration of uracil, deoxyuridine, uridine, thymine or thymidine. Uracil had more effect than other pyrimidine nucleotides in enhancing antitumor effect of these drug to FT-207 without toxicity. In contrast to the results with FT-207, co-administration of uracil with 5-FU increased its antitumor activity and also the toxicity of 5-FU. On the ther hand, no enhancing antitumor activity of dRib-1-P, Rib-1-P, Ado, Xao, Guo, Ino, dAdo, dIno and dGuo to FT-207 was observed. Concentration of 5-FU in the tumor, blood and various organs of AH-130-bearing rats after oral administration of clinical doses of FT-270 and uracil was examined. On oral administration of FT-207 plus uracil in various combinations, the highest T/B (ratio of concentration of 5-FU in the tumor to that in blood) value was obtained at a ratio of uracil to FT-207 of 4. Moreover, it was found that 5-FU was mainly phosphorylated in the tumor tissues whereas it was mainly degraded in liver slices. Degradation of 5-FU in vitro was inhibited more by thymine than by uracil. Phosphorylation of 5-FU, however, was not inhibited by uracil, thymine or thymidine, even times the concentration of 5-FU. These findings suggest that the enhancement of antitumor activity of 5-FU-masked form is more important the inhibition of 5-FU degradation than the stimulation of 5-FU phosphorylation.

Separation of cytidine diphosphate reductase from rat Yoshida ascites sarcoma.

CDP reductase was separated from the cytosol of rat Yoshida ascites sarcoma. The precipitate, which resulted from the acidification of the cytosol by acetic acid at pH 5.2, catalyzed specifically the reduction of CDP, whereas the concurrently resulted supernatant catalyzed those of UDP, ADP and GDP. The CDP reductase showed a single peak in the pattern of the enzyme activity in DEAE-cellulose and also in Sepharose 4B column chromatography with adequate recovery of the activity.

IMP dehydrogenase. I. Studies on regulatory properties of crude tissue extracts based on an improved assay method.

Inhibition of conversion from IMP to uric acid, which interferes with both spectrophotometric and radioisotopic assays of IMP dehydrogenase, by addition of allopurinol (0.1 mM), an inhibitor of xanthine oxidase, to the incubation system made it possible to determine the enzyme activity in crude liver extracts. With this improved assay method, the regulatory properties of the enzyme in crude extracts of liver and Yoshida sarcoma ascites cells were examined. In both tissues IMP dehydrogenase was found in the postmicrosomal supernatant. However, further centrifugation resulted in precipitation of the enzyme, the enzyme from Yoshida sarcoma ascites cells being precipitated more easily than that from rat liver. It was also found that IMP dehydrogenase activity increased during liver regeneration and that this increase was associated with the precipitate from the postmicrosomal fraction. These findings suggest that such a large sedimentable complex including IMP dehydrogenase might be formed in relation to cell growth. Most of the enzyme activity in rat liver and Yoshida sarcoma ascites cells was extracted in the supernatant obtained by centrifugation at 105,000 X g for 4 h after treatment of tissue homogenates with 1 M KCl, 0.75 M (NH4)2SO4, 2 M dimethylsulfoxide, 2 M KSCN, 25% glycerol, or 0.8 M guanidine-HCl. Treatment with 2% deoxycholate, 2% Triton X-100 or 2 M urea gave limited extraction. The enzyme was retained on a phenyl-Sepharose CL-6B or octyl-Sepharose CL-6B column and eluted with 0.8 M guanidine-HCl. These results suggested that the enzyme molecule has not only ionic but also hydrophobic domains, through which it interacts with other molecules of the enzyme itself and/or postmicrosomal cellular components.(ABSTRACT TRUNCATED AT 250 WORDS)

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells.

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)