Constituents of Plant Leaf Wax Contained in Rice Callus Tissues

Two enzyme preparations having both nuclease and 3′-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography.

The enzyme activities for RNA, native DNA, heat-denatured DNA and 3′-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymes were shown to be endonucleases (EC 3.1.30.2) which liberated 5′-mononucleotides and oligonucleotides from both RNA and DNA with the following relative rate of hydrolysis: RNA > native DNA = heat-denatured DNA.     

Identity of Phosphodiesterase and Phosphomonoesterase Activities with Nuclease P1 (a Nuclease from Penicillium citrinum)

Enzymatic properties of a purified Penicillium nuclease (designated as nuclease P₁) were investigated. The enzyme activities for RNA, heat-denatured DNA, native DNA, 3′-AMP and 2′-AMP showed a great degree of similarity with respect to the following properties: a) Range of stable pH (5~8), b) temperature optima (at around 70°C), c) thermostability (about 50% inactivation at 67°C, pH 6.0 for 15 min, d) effect of metal ions and SH inhibitors, e) requirement of Zn²⁺, f) protection from the heat-inactivation by albumin and Zn²⁺, g) inactivation on standing in the cold and reactivation on heating, h) sensitivity to protease, and i) competitive relationship between substrates in the enzyme reaction. Moreover, the ratio of enzyme activities in several mutants of Penicillium citrinum was constant. From these results, together with constant ratio of the specific activities throughout purification, it is concluded that a single enzyme might be responsible for both phosphodiesterase and phosphomonoesterase functions.     

Demethylamination of N6-methyldeoxyadenosine by commercial spleen phosphodiesterase preparations

Commercial spleen phosphodiesterase preparations contain an activity (presumably enzymatic) that will deaminate adenosine, deoxyadenosine and demethylaminate N⁶-methyldeoxyadenosine but not their 3′ -or 5′-phosphates. The product obtained by the enzymic demethylamination of N⁶-methyldeoxyadenosine was identified as deoxyinosine on the basis of its electrophoretic mobility and its ultraviolet spectrum. The presence of a deaminase activity in spleen phosphodiesterase preparations must be considered when using the enzyme in nucleotide sequence studies.     

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.     

Isolation of unselected mutants of alkaline phosphatase in Escherichia coli through nitrosoguanidine comutation and comparison with natural variants

We have devised a general procedure to isolate enzymatic variants without selecting or screening for related phenotypic peculiarities of the organism. A high mutation rate at phoA, the structural gene for alkaline phosphatase, is found among N-methyl-N'-nitro-N-nitrosoguanidine-induced proC revertants of Escherichia coli. About 1.6% of such revertants lack alkaline phosphatase, and many others exhibit altered enzyme parameters. Three mutants studied in detail had full enzyme activity but differed from the wild type in electrophoretic mobility, thermostability, and, in one case, optimum pH for enzyme activity. Four other phosphatase variants were discovered in a survey of 50 natural E. coli isolates; their electrophoretic mobility and thermostability were different from those of the wild type. Natural and induced enzyme variants are similar enough to suggest the absence of strong selective pressures in natural populations.This work was supported by grants from the Fundación J. March and the Comisión Asesora para la Investigación Científica y Técnica.     

Thermostability characteristics of glucosephosphate and triosephosphate isomerase in erythrocytes from several species

Significant differences in the thermostability of both glucosephosphate and triosephosphate isomerase were noted among a series of six primate and five nonprimate species. The enzyme structural differences among species, as assessed by thermostability profiling, was greater than expected from electrophoretic mobility patterns. Microheterogeneity of GPI, i.e. differences in thermostability within a species that are not detectable by electrophoresis, was detected in two primate species. Major differences in the levels of erythrocyte enzyme activity were observed with human and cow differing by 18-fold for TPI and baboon and cow differing by seven-fold in GPI activity.     

Biochemical, electrophoretic and immunological characterization of peroxisomal enzymes in three soybean organs

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti-cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20-fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Glucose 6-phosphate dehydrogenase in Drosophila: Sexual effects on structure

Glucose 6-phosphate dehydrogenase produced by female Drosophila melanogaster differs from that of males in electrophoretic mobility, kinetics, and thermostability. The enzyme produced by pseudomales is intermediate in kinetics and stability, but resembles that of males in electrophoretic mobility. Stability and kinetics are affected by growth temperature in pseudomales.This research was supported by NIH grants, Nos. 5-T1-GM 216-06 and GM 12768-01 and NSF grants GB 4587 and GB 4824.     

A comparative characterization of cytosolic superoxide dismutase/(SOD) from hog liver and erythrocytes.

1. Conditions of preparation and purification of superoxide dismutase from hog liver and erythrocytes were established. 2. The enzymes from both tissues were compared in respect to electrophoretic mobility, pI value, amino acid composition and spectrophotometric profiles and some differences were observed. 3. Conditions of enzyme dissociation were elaborated and molecular weights of subunits obtained from both kinds of SOD were found to be approx. 16,000. 4. Effect of heat and pH on the enzyme activity were tested. Both enzymes exhibited a relative thermostability.     

Enzydmatic Characterization of Babesia bovis1

ABSTRACT. Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.     

Effect of RNA and histone preparations on the properties of lymphoid cells

Preparations of RNA and histones change the electrophoretic mobility of lymphoid cells, the intensity of their respiration and anaerobic glycolysis, and thermo-merchanical properties of DNA-complexes isolated from lymphoid cells. RNA and histones exert their influence on the properties of DNP-systems and the rate of anaerobic glycolysis in the opposite manner. But both these preparations depress the electrophoretic mobility and oxygen consumption.     

Molecular nature of β-Galactosidase from different tissues in two strains of the house mouse

One inbred mouse strain, C57BL/Kl, has high galactosidase activities in all tissues while another strain, DBA/2/Kl, has low activities determined by the Bgs locus. Beta-Galactosidase from these two strains was partly purified by a five-step procedure: acidification, ammonium sulfate precipitation, gel filtration at two pHs, and isoelectric focusing. No qualitative differences were found between the enzyme preparations from the two strains. They had identical heat inactivation curves, pH optima, molecular weight, and isoelectric points, and the Km values were very similar. It thus seems that this genetic difference in enzyme activity probably cannot be explained by a variation of the galactosidase-specific activity but rather reflects a difference in number of enzyme molecules. Eight different isoenzymes were separated from liver, kidney, and spleen. Each isoenzyme has a different electrophoretic mobility and there is a stepwise increase in molecular weight from 143,000 to 380,000 beginning with the protein having the lowest isoelectric point. A likely interpretation is that the isoenzymes bind a smaller polypeptide in varying numbers in addition to the enzymatic polypeptide per se.     

The last two pathway-specific enzyme activities of hexosamine biosynthesis are present in Blastocladiella emersonii zoospores prior to germination

Forward direction assays have been developed for the last two pathway-specific enzymes of hexosamine biosynthesis using crude extracts from Blastocladiella emersonii zoospores. The specific enzyme activities measured are substantially higher than those reported with enzyme preparations from other organisms. During the development of one of the assays, another enzyme activity was observed which converts one of the intermediates of the pathway, N-acetylglucosamine-6-phosphate, to N-acetylglucosamine. The finding of these three enzyme activities in zoospore extracts completes the demonstration that all the enzyme activities necessary to synthesize some 2% by weight as chitin early during zoospore germination (de novo cell wall formation) pre-exist in the zoospore. This demonstration is consistent with the conclusion that the hexosamine pathway is regulated at the post-translational level during zoospore germination.     

A mutant of escherichia coli defective in removing 3′ terminal nucleotides from some transfer RNA precursor molecules

The conversion of precursor RNA into bacteriophage T4 proline and serine transfer RNAs includes two steps for the enzymatic removal of nucleotides from the 3′ ends of RNA chains. Neither of these steps occur following infection of a mutant of Escherichia coli that was previously shown to block the suppressor function of T4 serine transfer RNA. Cell-free extracts of this mutant are furthermore deficient in a wild type enzyme activity that removes nucleotides from the 3′ ends of one of the RNA chains described above. The relation of this enzyme to other 3′ ribonucleases is not known.We subsequently examined the mutant for its ability to support the biosynthesis of other bacteriophage transfer RNAs. In one instance that is analogous to the proline-serine precursor RNA, maturation of the precursor RNA was blocked during infection of mutant cells. In another instance, precursor RNA maturation was normal, even though this involved the removal of 3′ nucleotides. These observations point to the possible existence of at least two 3′ ribonucleases for the biosynthesis of transfer RNAs.     

The multiple forms and kinetic properties of the N-acetyl-beta-D-hexosaminidases from colonic tumours and mucosa of rats treated with 1,2-dimethylhydrazine.

The separation and purification of the N-acetyl-beta-D-hexosaminidase activities from tumours induced by 1,2-dimethylhydrazine in the rat colon and from colonic mucosa of tumour-bearing animals are reported. Mucosa contained N-acetylhexosaminidases A and B, as well as a third form whose properties with regard to electrophoretic mobility and thermostability lay between those of A and B. Tumours contained only N-acetylhexosaminidase A and B activities. Each form possessed both N-acetylglucosaminidase (EC 3.2.1.30) and N-acetylgalactosaminidase (EC 3.2.1.53) activities, which could not be separated by a variety of techniques. The alteration of the ratio of the two specific activities in each form during purification, together with differences in the kinetic inhibition constants and behaviour during inactivation by various reagents or a temperature of 50 degrees C, supported the belief that each form contains the two enzyme activities, glucosaminidase and galactosaminidase, at separate active sites. This model is in contrast with that reported for these activities from a number of other sources. A variety of treatments reported to cause the conversion of form A into a form resembling B failed to produce such an effect on the rat colonic hexosaminidases.     

Purification of a Nuclease from Penicillium citrinum

A nuclease was purified about 1500-fold with a recovery of 20% from an aqueous extract of culture of a pigmentless mutant VI–10–14 of Penicillium citrinum on wheat bran. The purified preparation was homogeneous on the basis of the criteria of ultracentrifugation and disc gel electrophoresis. The preparation was essentially free of 5′-nucleotidase, non-specific phosphomonoesterase, non-specific phosphodiesterase and 3′-monoester forming nuclease. The preparation hydrolyzed phosphodiester bonds in RNA and DNA to yield 5′-mononucleotides, and also the phosphomonoester bond in 2′- and 3′-AMP to yield nucleoside and inorganic phosphate. The enzyme activities toward these substrates were not separated and relative ratio of their specific activities remained constant throughout the purification, suggesting that a single enzyme was responsible for these activities.     

Purine and Pyrimidine 5′-Nucleotide Catabolism in Tetrahymena pyriformis W1

SYNOPSIS Deamination at pH 7.5 of adenosine, deoxyadenosine, cytidine and deoxycytidine by cell-free preparations of Tetrahymena pyriformis W was observed both in the presence and absence of fluoride. Deamination of 5′-AMP, 5′-dAMP, 5′-CMP, and 5′-dCMP was found only in the absence of fluoride. Dephosphorylation of the above nucleotides by acid phosphatases occurred at pH 4.5; reduced activity was noted at pH 7.5. Fluoride effectively blocked acid phosphatase activity at both pH values. This correlation of phosphatase and deaminase activities suggests a catabolic pathway for 5′-AMP and 5′-CMP whereby dephosphorylation precedes deamination. Radiolabelled substrates were used to test this hypothesis. The experiments were designed so that conversion of as little at 1.0% of the radiolabelled substrate to the deaminated product could be detected. No 5′-IMP or 5′-UMP, the expected deamination products of 5′-AMP and 5′-CMP, respectively, was recovered after incubation of the radiolabelled substrates with cell-free enzyme preparations. Thus, it appears that Tetrahymena has no 5′-AMP or 5′-CMP deaminases and that these compounds are deaminated only after conversion to nucleosides. Acid phosphatase activity toward 5′-GMP, 5′-dGMP, 5′-TMP, 5′-UMP, and 5′-XMP was also found.     

Changes of intestinal alkaline phosphatase produced by cholecalciferol or 1,25-dihydroxyvitamin D3 in vitamin D-deficient chicks

Treatment with cholecalciferol or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increases activity and changes electrophoretic mobility of alkaline phosphatase (alkPase) from duodenal brush border of vitamin D-deprived chicks. Three of the four molecular forms of the enzyme show reduced velocity of migration 9 h after 1,25(OH)2D3 or 24 h after vitamin D3. This change is reversed about 48 h later, when mobility of those bands is higher than that of controls. Incubation of enzyme preparations with exogenous neuraminidase produces the same electrophoretic modifications observed during the early stage, indicating that they are due to desialylation. Cholecalciferol or 1,25(OH)2D3 increase sialidase activity of duodenal brush border. This increment precedes that of alkPase and could account for the initial desialylation and moderate rise of alkPase. Cycloheximide markedly reduces alkPase in rachitic chicks and blocks the increase of the enzyme activity produced by vitamin D3, but does not modify the rise of sialidase or the reduction of alkPase electrophoretic mobility. The bimodal response of alkPase to 1,25(OH)2D3 or cholecalciferol comprises two different mechanisms: during a first stage, epigenetic modifications of preexisting enzyme can be triggered by the increased Ca2+ levels; in a second phase, there is activation of enzyme synthesis.