DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers

DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly α-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only β-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase.     

Relationship of CIF, LT, and PIF released in vitro by activated human lymphocytes. II. A further functional comparison of LT and PIF activities on HeLa and L-929 target cells.

Lymphotoxin (LT), and proliferation inhibition factor (PIF) activities found in 5-day supernatants of mitogen-activated human lymphocytes (SAL) were further compared. In agreement with previous results, the activities could not be distinguished functionally. Quantitative differences in the amount of activity detected in the SAL could be accounted for on the basis of target cell differences, concentration of the lymphocyte effector molecules in the supernatant, and the parameter employed to assess cell function. Growth inhibitory activity detected at high supernatant dilutions was completely reversible, whereas the cytotoxic activity detected at low supernatant dilutions was irreversible. When the active medium was fractionated on DEAE, two peaks of inhibitory activity were detected. Depending upon the amount of activity and target cell, both peaks of activity were growth inhibitory or cytotoxic. Since both peaks of material affected HeLa and L-929 cells, the materials were not species specific. Thus, it appears that cloning inhibitor factor, LT, and PIF activities may actually be measures of the same stable materials found in 5-day activated lymphocyte supernatants.     

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.     

Activity of pyrimidine degradation enzymes in normal tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and beta-ureidopropionase (beta-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of beta-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and beta-UP were also present in a variety of other tissues, especially in bronchus.     

Purine metabolism in Acholeplasma laidlawii B: novel PPi-dependent nucleoside kinase activity

Acholeplasma laidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion. Phosphoribosyltransferase activities for adenine, guanine, xanthine, and hypoxanthine were shown. Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside. Adenosine, inosine, xanthosine, and guanosine were converted to their base. No ATP-dependent phosphorylation of nucleosides to mononucleotides was found. However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected. Nucleotidase activity for AMP, inosine monophosphate, xanthosine monophosphate, and GMP was also found. Interconversion of GMP to AMP was detected. Enzyme activities for the interconversion of AMP to GMP were not detected. Therefore, A. laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates. De novo synthesis of purines was not detected. This study demonstrates that A. laidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp. mycoides. This is the first report of a PPi-dependent nucleoside kinase activity in any organism.     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。     

Enzymatic repair of premutagenic DNA lesions in human epidermis. Quantitation of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities

Extracts of human epidermis prepared by the suction blister method were used to measure O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities. Although both activities were detected in all extracts examined, a 4-5-fold interindividual variation in activity was found. No obvious correlation of the two enzyme activities with the age of the patient was observed. Neither was there any correlation between the level of uracil-DNA glycosylase activity and O6-methylguanine-DNA methyltransferase activity.     

Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays

Proteolytic activities in soluble protein extracts from Mamestra brassicae (cabbage moth) larval midgut were analysed using specific peptide substrates and proteinase inhibitors. Serine proteinases were the major activities detected, with chymotrypsin-like and trypsin-like activities being responsible for approximately 62% and 19% of the total proteolytic activity towards a non-specific protein substrate. Only small amounts of elastase-like activities could be detected. The serine proteinases were active across the pH range 7-12.5, with both trypsin-like and chymotrypsin-like activities maximal at pH 11.5. The digestive proteinases were stable to the alkaline environment of the lepidopteran gut over the timescale of passage of food through the gut, with 50% of trypsin and 40% of chymotrypsin activity remaining after 6h at pH 12, 37 degrees C. Soybean Kunitz trypsin inhibitor (SKTI) ingestion by the larvae had a growth-inhibitory effect, and induced inhibitor-insensitive trypsin-like activity. Qualitative and quantitative changes in proteinase activity bands after gel electrophoresis of gut extracts were evident in SKTI-fed larvae when compared with controls, with increases in levels of most bands, appearance of new bands, and a decrease in the major proteinase band present in extracts from control insects.     

Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. In pigeon liver, only purine nucleoside phosphorylase was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.     

Phenol oxidase activity in bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis>

Phenol oxidase activity was detected for the first time in a number of strains belonging to various Azospirillum species. Both extracellular and intracellular activities of laccase, Mn-peroxidase, lignin peroxidase, and tyrosinase were observed. Extracellular enzymes were found to have higher activity. Significant differences in phenol oxidase activities were observed between species and strains.     

Activity of Pyrimidine Degradation Enzymes in Normal Tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and ß-ureidopropionase (ß-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of ß-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and ß-UP were also present in a variety of other tissues, especially in bronchus.     

Comparative study of acid and alkaline phosphatase during the autolysis of filamentous fungi

Acid and alkaline phosphatase activities in culture liquid and mycelial extract during autolysis were studied in seven fungi of the general Ascomycotina, Basidiomycotina and Zygomycotina. High activities of extracellular and mycelial extract acid phosphatase and lower activities of alkaline phosphatase were found in Ascomycotina, and acid phosphatase was present in Basidiomycotina. In Zygomycotina only mycelial extract alkaline phosphatase activity was detected. A correlation between degree of autolysis, pH and acid phosphatase activity was demonstrated.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

Mannitol oxidation in two Micromonospora isolates and in representative species of other actinomycetes.

Mannitol kinase and mannitol-1-phosphate dehydrogenase activities were detected in two Micromonospora isolates. The presence of these enzyme activities indicates that mannitol is catabolized first to mannitol-1-phosphate and then to fructose-6-phosphate. Mannitol-oxidizing enzymes were also surveyed in representative species of four other genera of actinomycetes. Mannitol-1-phosphate dehydrogenase was detected in cell-free extracts of Streptomyces lactamdurans. In contrast, cell-free extracts of Mycobacterium smegmatis, Nocardia erythrophila, Streptomyces lavendulae, and Actinoplanes missouriensis contained mannitol dehydrogenase activity but no detectable mannitol-1-phosphate dehydrogenase activity. The mannitol dehydrogenase activities in the latter species support the operation of a pathway for catabolism of mannitol that involves the oxidation of mannitol to fructose, followed by phosphorylation to fructose-6-phosphate.     

Mannitol oxidation in two Micromonospora isolates and in representative species of other actinomycetes.

Mannitol kinase and mannitol-1-phosphate dehydrogenase activities were detected in two Micromonospora isolates. The presence of these enzyme activities indicates that mannitol is catabolized first to mannitol-1-phosphate and then to fructose-6-phosphate. Mannitol-oxidizing enzymes were also surveyed in representative species of four other genera of actinomycetes. Mannitol-1-phosphate dehydrogenase was detected in cell-free extracts of Streptomyces lactamdurans. In contrast, cell-free extracts of Mycobacterium smegmatis, Nocardia erythrophila, Streptomyces lavendulae, and Actinoplanes missouriensis contained mannitol dehydrogenase activity but no detectable mannitol-1-phosphate dehydrogenase activity. The mannitol dehydrogenase activities in the latter species support the operation of a pathway for catabolism of mannitol that involves the oxidation of mannitol to fructose, followed by phosphorylation to fructose-6-phosphate.     

Induction and quantification of phenylacyl-CoA ligase enzyme activities in Pseudomonas putida CA-3 grown on aromatic carboxylic acids

Three phenylacyl-CoA ligase activities were detected in extracts of Pseudomonas putida CA-3 cells grown with a variety of aromatic carboxylic acids. The three phenylacyl-CoA enzyme activities measured were phenylpropyl-CoA ligase (acting on both phenylpropanoic acid and cinnamic acid), a phenylacetyl-CoA ligase, and a medium chain length phenylalkanoyl-CoA ligase acting on aromatic substrates with 5 or more carbons in the acyl moiety. The rate of each enzyme activity detected in extracts of P. putida CA-3 cells is dependent on the growth substrate supplied. High rates of phenylpropyl-CoA ligase activity were observed with extracts of cells grown on phenylpropanoic acid, cinnamic acid or medium chain length phenylalkanoic acids with an uneven number of carbons in the acyl moiety. Extracts of P. putida CA-3 cells exhibited high rates of phenylacetyl-CoA ligase activity when grown on phenylacetic acid or medium chain length phenylalkanoic acids with an even number of carbons in the acyl moiety. In addition, high rates of medium chain length phenylalkanoyl-CoA ligase activity, towards phenylvaleric acid and phenylhexanoic acid, were exhibited by extracts of cells grown on all medium chain length phenylalkanoic acids. Low levels of the various phenylacyl-CoA ligase activities were found in extracts of cells grown on benzoic acid and glucose. Benzoyl-CoA ligase activity was not detected in any cell free extracts generated in this study.     

Diversity of L-methionine catabolism pathways in cheese-ripening bacteria

Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum. The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.     

Salvage synthesis of purine nucleotides by Helicobacter pylori

G.L. MENDZ, B.M. JIMENEZ, S.L. HAZELL, A.M. GERO AND W.J. O'SULLIVAN. 1994. The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.     

Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

Cholinesterase and acid phosphatase (AP), but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.