Two forms of glucoamylase (GI and GII) from starch-grown Lipomyces kononenkoae CBS 5608 mutant were purified to apparent homogeneity by means of ultrafiltration, Sephacryl S-200 gel filtration and DEAE Sephadex A-50 chromatography. The apparent molecular weight was calculated as ca. 150 kDa for GI and ca. 128 kDa for GII, respectively. Both enzymes were glycoproteins with isoelectric points of 5.6 (GI) and 5.4 (GII). They had a pH optimun of 4.5 and were stable from pH 5 to 8. The temperature optimum for both enzymes was , but they were rapidly inactivated above . The values toward starch were estimated to be 6.57 mg per ml for GI and 4.52 mg per ml for GII, and the values were 16.28 per mg for GI and 32.25 per mg for GII, respectively. The and values of GII for or were estimated to be 0.15 mg per ml and 2.0 mg per ml, respectively () and 1.02 per mg or 1.02 per mg, respectively (). Neither enzyme exhibited pullulanase activity but they released only glucose from starch or cyclodextrin. Amino acid analysis indicated that both glucoamylases were enriched in proline and acid amino acids. Glucoamylase GII strongly cross-reacted with a monoclonal antibody raised against GI enzymes, and the two enzymes shared very similar amino acid composition. Western blot analysis indicated that L. kononenkoae CBS 5608 mutant produced two forms of glucoamylase on starch, and that synthesis of them was subject to glucose repression.

One of the reaction pathways in light-invoked signal transduction can be initiated through ion fluxes across the plasma membrane in higher plants. We isolated protoplasts from oat coleoptile and examined the effects of light on the membrane potential using a membrane potential-sensitive fluorescent probe (bisoxonol). Both red and far-red light initially induced a hyperpolarization in oat cells. Red light-induced hyperpolarization was effectively dissipated by 100 mM , but the hyperpolarization induced by far-red light was not depolarized by any of the cations (, , , ) tested. The depolarization induced by red light and was inhibited by 200 mM TEA, which is a channel blocker. These results suggest that influx through the inward channel may be a depolarization path in the phytochrome-mediated signal transduction.

In order to investigate whether phospholipase C (PLC) activity in oat celIs is regulated by Gprotein, we have characterized PLC in plasma membranes of oat tissues. To identify the purified plasma membrane, -stimulated, -dependent ATPase activity was measured. The activity of ATPase was shown to be proportional to the concentration of membrane protein. To examine the PLC activity regulated by G-protein, we used the inside-out and outside-out plasma membrane mixture isolated from the oat cells. The plasma membrane mixture showed higher PLC activity than the one of the outside-out plasma membrane. This suggests that PLC activity is located at the cytoplasmic surface of plasma membrane. PLC activity in plasma membrane mixture was dependent on with maximum activity at 100 and it was inhibited by 1 mM EGTA. Using Sep-pak Plus QMA chromatography, we found that inositol 1,4,5-trisphosphate () was produced in the presence of 10 . The PLC activity in the membrane was enhanced by an activator of G-protein () and not by an inhibitor (). This indicates that a G-protein is involved in the activation of PLC in the plasma membrane of oat cells.

The effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the intracellular level was studied in NIH3T3 fibroblast cells. A reduction of the intracellular level was observed after exposure to 300 MNNG. However, the intracellular level of , a well-known regulator of release from internal storage, was not changed by MNNG treatment. Instead, a reduction of the intracellular NAD level was observed. NAD as well as stimulated intracellular release from permeabilized cells. The treatment of 3-aminobenzamide, which inhibited the MNNG-induced reduction of the NAD level, also prevented the MNNG-induced decrease of the level. Our data suggest that MNNG reduces the intracellular level by NAD depletion in NIH3T3 cells.

Two fibrinolytic enzymes from the autolysate of Lumbricus rubellus were purified in homogeneous form. Their molecular sizes were 31,000 (Enz1) and 35,000 (Enz2) Da. respectively. However, the N-Terminal amino acid sequences of Enz1 and Enz2 were exactly the same: Ile-Val-Gly-Gly-Ile-Glu-Ala-Arg-Pro-Tyr-Glu-Phe-Pro-Trp-Gln-. These results indicate that Enz1 is a shortened form of Enz2 formed during autolysis. When a synthetic substrate, Ile-Pro-Arg-pNA, was used, the catalytic activity were observed in the pH range of 5-10 and the kinetic parameters including (1.6 ) and (40 nmol/jmin/mg) were almost identical between the two enzymes. However, the fibrinolytic activity of Enz2 was at least 1.25 times higher than that of Enz1, suggesting that the C-terminal region of Enz2 is important in fibrinolysis but not in amidolysis. Furtheimore. fibrinolytic activity of the enzymes was increased by the addition of the lipid extracted from L. rubellus in the presence of or . The stimulatary effect of lipid on Enz2 was higher compared to Enz1.

Single-run Partial cDNA sequencing was conducted on 1,592 randomly selected human fetal liver cDNA clones of Korean origin to isolate novel genes related to liver functions. Each partial cDNA sequence determined was analyzed by comparing it with the databases. GenBank, Protein Information Resource (PIR) and SWISS-PROT Protein Sequence Data Bank. From a set of 1.592 cDNA clones reported here, 1,433 (90.0% of the total) were informative cDNA sequences. The other 159 clones were identified as DNA sequences which had originated from the cloning vector. Among 1,433 informative partial cDNA sequences, 851 (59.3%) clones were revealed to be identical to known human genes. These known genes have been classified into 225 different kinds of genes. In addition, 340 clones (23.7%) showed various degrees of homology to previously known human genes. Ninety four (6.6%) clones contained various repeated sequences. Twenty four (1.7%) partial cDNA sequences were found to have considerable homology to known genes from evolutionarily distant organism such as yeast, rice, Arabidopsis, mouse and rat, based on database matches, whereas 124 (8.7%) had no Significant matches. Human homologues to functionally characterized genes from different organisms could be classified as candidates for novel human genes of similar functions. Information from the partial cDNA sequences in this study may facilitate the analysis of genes expressed in human fetal liver.

Decrease in the amount of cotyledonary spermidine in Glycine max under anaerobic conditions related to an increase in spermidine dehydrogenase. Under the same conditions, no enzymatic activity of diamine oxidase was observed. Exposure of Glycine max both to spermidine and 1,3-diaminopropane under anaerobic conditions resulted in a decrease in spermidine contents. Correlated with the decrease in spermidine contents, there was a drastic increase in spermidine dehydrogenase. The molecular weight of the purified enzyme estimated by Sephacryl S-300 gel column and SDS gel electrophoresis were 130,000 dalton and 65,000 dalton, respectively, indicating that the enzyme is a dimer. The optimal pH for activity was 9.3. The value for spermidine was 0.61 mM. Neither metal ions nor polyamine and derivatives affected enzymatic activity, but the enzyme was inhibited by DTNB, NEM and PCMB, suggesting that a cysteine residue of the enzyme is associated with or involved in enzyme activity. To our knowledge, this is the first report describing properties of the enzyme from plants. Considered together, the data in this paper indicate that both spermidine and 1,3-diaminopropane, novel activators, enhance the spermidine dehydrogenase activity and control the intracellular spermidine contents.

The retroviruses carrying and oncogenes were infected into fetal thymic organ culture (FTOC) to study the molecular mechanisms involved in T cell development. T cell lymphomas in the different stages of T cell development were obtained from this culture system. Interestingly, a few cell lines obtained from this system have a lack of transfected oncogenes, however these cells have the characteristics of transformed cells. In spite of the discrete phenotype of these transformed cell lines, the same pattern of recombination of endogenous c-raf genes was detected from Southern blot analysis. We suggest in this regard that the translocation event of thymocytes, or abnormal promoter activity, can cause lymphomagenesis by way of c-raf.

This study was conducted to compare the effects of n-6 linoleic acid and n-3 linolenic acid on lipid peroxidation and the activities of enzymes defending against oxidation, which are involved in the tumor promotion, and histolOgical changes of hepatocarcinogen treated rat liver. In this study, weanling male Sprague-Dawley rats were fed one of three diets, containing 15% (w/w) of beef fat (BF), com oil (CO) or perilla oil (PO), for 11 weeks. During the 3rd week, experimental groups were injected with 2-AAF (50 mg/kg of BW) intraperitoneally 3 times. Findings show that the com oil diet group has greater liver MDA content than the beef fat and perilla oil diet groups. Also, it is observed that the perilla oil diet group has lower MDA content than beef fat and com oil diet groups, even though perilla oil is more desaturated than beef fat and com oil. In terms of activity, mixed-function oxidase activity is not Significantly affected by the different dietary fats and 2-AAF treatment. GSH-peroxidase, GSH-reductase and GSH-Stransferase activities are significantly higher in the CO+AAF group than those of the other groups. GST and GSH-Px are activated by 2-AAF treatment in the com oil diet group only. The hepatocytes of the BF+AAF group were the most severely degenerated, the second was the CO+AAF group and the least was the PO+AAF group. It was also found that dietary com oil increased lipid peroxidation and activated defense enzymes against oxidation in liver, but dietary perilla oil did not, or supressed defense enzymes. Therefore it is concluded that dietary n-3 linolenic acid in perilla oil inhibits lipid peroxidation and carcinoenesis in rat liver following 2-AAF treatment.

Ganglioside GM3 and sialidase activities in human fetal liver have been investigated. Gangliosides were extracted from fetal livers by the Folch-Suzuki method and analyzed by high-performance thin layer chromatography (HPTLC). GM3 increased, but lactosylceramide (LacCer) decreased predominantly over the developmental stages. Sialidase in human fetal liver was mainly localized in the lysosomal fraction and its activity was high in the earlier stages of development. The optimum pH for this enzyme was 4.3~4.4. Sialidase was more active with the ganglioside mixture than with GM3, sialyllactose or fetuin. Fetal liver sialidase was still active (20% activity) in the presence of 25% methanol. These results suggested that the changes of the ganglioside GM3 and sialidase activity may be involved in the regulation of cell growth in human fetal liver during development.

In order to assign NMR signals, conditions for the specific labeling of cytochrome of D. vulgaris Miyazaki F through the culture in a minimal medium were established. Phenylalanine residue was specifically deuterated at more than 85% efficiency. Cytochrome has two phenylalanine residues. The signals of one phenylalane were missing and this was tentatively assigned to Phe20.

The potency of yeast-derived methionyl-free human growth hormone (rhGH), which was obtained by removal of the N-terminal Met from methionyl-hGH, was estimated by in vitro and in vivo assays. In radio-receptor assay where the binding affinity of growth hormone to the receptor was estimated, the recombinant hGH showed 2.9 international units (IU) per mg of specific activity. In contrast, pitUitary-derived human growth hormone had a slightly lower receptor binding activity (2.5 IU/mg) compared with recombinant growth hormone. For the in vivo assay, efficacy of rhGH was tested by use of hypophysectomized rats, in which pituitary organs were surgically removed, resulting in the termination of growth hormone secretion. The weight-increase in rats by the injection of rhGH was almost identical to the result obtained by the injection of the same amount of pituitary-derived (international standard) hGH. A comparision of the secondary structures of rhGH and rMet-hGH by circular dichroism spectrophotometer demonstrated that the removal of the methionyl residue from rMet-hGH did not exert any effect on the structure of the growth hormone. In conclusion, methionyl-free human growth hormone produced from yeast was highly potent in biological activity and maintained a legitimate three dimensional structure.

Cloned staphylococcal peptidoglycan hydrolase was used in determining the physiological characteristics of peptidoglycan hydrolase. This enzyme hydrolyzed the bacterial cell walls and released the N-terminal alanine, but not the reducing groups. This cloned gene product was localized in the cytoplasm of transformed Escherichia coli. Activity gels indicated the enzyme had an Mr of about 54,000, which was consistent with the deduced Mr from sequencing of the cloned gene. The activity bound to CM-cellulose but not DEAE-cellulose resin, indicating it as a basic protein. Enhanced enzyme activity in a low concentration of cations, and inhibited enzyme activity in a solution with dissolved phospholipids, suggested that the activity and the availability of this basic protein may be regulated between negatively charged and positively charged cellular molecules. The activity against boiled crude cell wall was much greater than against purifed cell wall, suggesting protein associated with crude cell wall may aid in the binding of the peptidoglycan hydrolase The cloned peptidoglycan hydrolase showed positive activity on whole cells of some lysostaphin-resistant coagulase-negative staphylococci. The cloned enzyme may be an alternative for lysostaphin for lysis of staphylococci.

Four carboxypeptidases, CP1, CP2, CP3, and CP4 were isolated from the cotyledons of germinating seedlings of Canavalia lineata by sequential chromatography on the following four columns: 1) CM-cellulose, 2) Sephacryl 5-300, 3) Procion red dye, and 4) Sephacryl S-200. A number of properties of the enzymes, such as substrate specificity, molecular weight, optimum pH, thermal stability, have been determined. Enzyme activities were measured using the Cbz(carbobenzoxy)-dipeptides containing phenylalanine at the penultimate position. The values of four carboxypeptidases for Cbz-Phe-Ala were 0.50, 0.65, 1.30, and 1.35 mM, respectively. The inhibition studies indicated that the four carboxypeptidases were all serine type. Each of the carboxypeptidases with molecular weights of 145, 114, 105, and 104 kDa, respectively, had the optimum enzyme activity at pH 5.0~6.0. And they were sensitive to high temperature.

Two apparent rho-independent transcription terminator structures within the coding sequence of aceK have been destroyed to access their roles in the differential expression between aceA and aceK in the glyoxylate bypass operon of E. coli. The effect of mutations on the expression of aceK was evaluated in two different ways: one by maxicell labeling and the other by lacZ fusion gene construction. The maxicell labeling experiment with the mutant operon clones has failed, like that of the wild type operon clone, to visibly show isocitrate dehrogenase (IDH) kinase/phosphatase, the product of aceK, on the autoradiogram of a protein gel. When the same mutations were introduced into an aceK::lacZ fusion gene to quantitatively evaluate the mutational effect, the activity of in neither of the mutant versions of the fusion gene was elevated significantly enough to explain the degree of polarity observed in this region. Thus, we conclude that neither of these intragenic, apparent rho-independent transcription terminator structures, which have long been suspected as a major determinant in the down regulation of aceK, really act as a premature transcriptional terminator.

Quinolone antibiotics are DNA gyrase inhibitors, but their bactericidal action seems to involve more than the inhibition of DNA gyrase activity. Hence, the potentially crucial factors among possible mechanisms of quinolone action; cleavable complex formation, inhibition of DNA synthesis, and induction of SOS response were investigated. These parameters were measured in an Escherichia coli strain exposed to quinolones in the logarithmic growth phase, and correlated with the bactericidal activity of quinolones. Cleavable complex formation proved to be the factor most related to bactericidal action. Inhibition of DNA synthesis was substantially correlated with bactericidal activity, but induction of SOS response was least correlated with bactericidal activity. Therefore, it was concluded that quinolones exert bactericidal action primarily through cleavable complex formation, and subsequent unknown cellular processes together with inhibition of DNA synthesis contribute to the bactericidal activity of quinolones.