A CHARACTERIZATION OF NUCLEOSIDE DIPHOSPHATASE IN THE ONION ROOT TIP

1973 ◽  
Vol 21 (5) ◽  
pp. 417-425 ◽  
Author(s):  
W. D. KLOHS ◽  
C. W. GOFF
Keyword(s):  
Enzyme Activity ◽  
Lead Nitrate ◽  
Maximal Activity ◽  
Root Tip ◽  
Uridine Diphosphate ◽  
Onion Root ◽  
Guanosine Diphosphate ◽  
Inosine Diphosphatase ◽  
Triton X ◽  
Nucleoside Diphosphatase

Inosine diphosphatase of a Golgi-enriched fraction of the onion root tip was characterized. Peak enzyme activity occurred at pH 4.8 and 7.0, although considerable activity was present between the peaks. The activity at neutral pH approximately doubled during a 4-day cold storage; no such increase of the pH 4.8 activity occurred under similar conditions. Treatment with deoxycholate or Triton X-l00 also activated the pH 7.0 enzyme. Although magnesium, manganese and calcium all supported inosine diphosphatase activity, 2 mM manganese supported maximal activity. Uridine diphosphate, guanosine diphosphate and inosine diphosphate were hydrolyzed much more rapidly than the other substrates tested. Sodium fluoride, uranyl nitrate, potassium chloride and heat all partially inhibited the enzyme. Glutaraldehyde and lead nitrate, two reagents to which nucleoside diphosphatase is exposed when studied cytochemically, greatly reduced enzyme activity.

scholarly journals Nucleoside diphosphatase in the onion root tip. II. The effect of inhibitors on isoenzymes.

1977 ◽  
Vol 25 (11) ◽  
pp. 1247-1253 ◽  
Author(s):  
G D Troyer ◽  
C W Goff ◽  
W D Klohs
Keyword(s):  
Adenosine Diphosphate ◽  
Guanosine Monophosphate ◽  
Thiamine Pyrophosphate ◽  
Root Tip ◽  
Root Extract ◽  
Uridine Diphosphate ◽  
Electrophoretic Study ◽  
Onion Root ◽  
Cytidine Diphosphate ◽  
Nucleoside Diphosphatase

An electrophoretic study was performed to determine the number of isoenzymes of nucleoside diphosphatase in onion root extract. Five bands exhibiting nucleoside diphosphatase activity were detected when gels were incubated with inosine diphosphate, uridine diphosphate, guanosine diphosphate or cytidine diphosphate as substrates. These consisted of a single fast migrating band (band one), a group of three intermediate migrating bands (bands two, three and four) and a single slow migrating band (band five). Gels incubated with adenosine diphosphate, thiamine pyrophosphate, inosine monophosphate, guanosine monophosphate and cytidine monophosphate showed only two bands (bands one and five). Inhibitor studies showed that sodium fluoride inhibited bands one and five but not bands two, three and four. Conversely, 1% (v/v) glutaraldehyde inhibited bands two, three and four but did not inhibit bands one and five. These results suggest that two separate groups of onion nucleoside diphosphatase isoenzymes occur which have different substrate specificities and are selected against by certain inhibitors.

scholarly journals NUCLEOSIDE DIPHOSPHATASE IN THE ONION ROOT TIP I. EFFECTS OF FIXATION AND LEAD ON ENZYME ACTIVITY

1974 ◽  
Vol 22 (10) ◽  
pp. 945-951 ◽  
Author(s):  
C. W. GOFF ◽  
W. D. KLOHS
Keyword(s):  
Enzyme Activity ◽  
Root Tip ◽  
Fixation Time ◽  
Onion Root ◽  
Nucleoside Diphosphatase

The terminal 0.2—0.5 mm of 3-day onion roots grown from bulbs were excised and fixed for 1½ hr in various concentrations of cacodylate-buffered glutaraldehyde, formaldehyde or combinations of these. Alternatively, the fixative concentration was held constant while fixation time was varied. Control roots were run in buffer lacking fixative. The roots were then homogenized and in most cases aliquots of the entire homogenate were used to assay for total nucleoside diphosphatase (NDPase). Parallel assays were usually run after treating the homogenate with deoxycholate. Both glutaraldehyde and formaldehyde inhibited NDPase activity, the extent of this inhibition depending upon fixation time, fixative concentration and the particular aldehyde used, although with both fixatives inhibition did not increase beyond a certain level even with further increase in fixation time or fixative concentration. With glutaraldehyde and glutaraldehyde-containing fixatives, this level was normally about 74% inhibition while with formaldehyde it was about the level of non-activated NDPase activity and inhibition could be detected only after deoxycholate treatment. Lead did not appear to inhibit the "fixed" NDPase.

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

1978 ◽  
Vol 26 (10) ◽  
pp. 772-781 ◽  
Author(s):  
W D Klohs ◽  
C W Goff ◽  
R J Bernacki
Keyword(s):  
Enzyme Activity ◽  
Lead Nitrate ◽  
Initial Step ◽  
Lead Ions ◽  
Fixation Time ◽  
Animal Tissues ◽  
Root Tips ◽  
Cytochemical Localization ◽  
Onion Root ◽  
L1210 Cells

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.

scholarly journals The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1970 ◽  
Vol 117 (2) ◽  
pp. 319-324 ◽  
Author(s):  
G. J. Mulder
Keyword(s):  
Enzyme Activity ◽  
Density Gradient ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Male Rats ◽  
Uridine Diphosphate ◽  
Triton X

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The Km of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.

scholarly journals Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.

1983 ◽  
Vol 97 (2) ◽  
pp. 340-350 ◽  
Author(s):  
D Godelaine ◽  
H Beaufay ◽  
M Wibo ◽  
A M Ravoet
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Protein Glycosylation ◽  
Permeability Barrier ◽  
Endoplasmic Reticulum Membrane ◽  
Uridine Diphosphate ◽  
Guanosine Diphosphate ◽  
Membrane Barrier ◽  
Dolichol Pathway ◽  
Triton X

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

Localization of nucleoside diphosphatase in the onion root tip

PROTOPLASMA ◽  
1973 ◽  
Vol 78 (4) ◽  
pp. 397-416 ◽  
Author(s):  
Charles W. Goff
Keyword(s):  
Root Tip ◽  
Onion Root ◽  
Nucleoside Diphosphatase

scholarly journals Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

1977 ◽  
Vol 75 (1) ◽  
pp. 135-147 ◽  
Author(s):  
A L Blitz ◽  
R E Fine ◽  
P A Toselli
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Coated Vesicles ◽  
Potassium Oxalate ◽  
Triton X ◽  
Trapping Agent ◽  
The Brain

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals Effects of Maytenus ilicifolia Mart. and Bauhinia candicans Benth infusions on onion root-tip and rat bone-marrow cells

2002 ◽  
Vol 25 (1) ◽  
pp. 85-89 ◽  
Author(s):  
Marjori Leiva Camparoto ◽  
Rosangela de Oliveira Teixeira ◽  
Mário Sérgio Mantovani ◽  
Veronica Elisa Pimenta Vicentini
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Root Tip ◽  
Onion Root ◽  
Maytenus Ilicifolia ◽  
Rat Bone ◽  
Marrow Cells

scholarly journals Altered sexual differentiation of hepatic uridine diphosphate glucuronyltransferase by neonatal hormone treatment in rats

1979 ◽  
Vol 180 (2) ◽  
pp. 313-318 ◽  
Author(s):  
Coral A. Lamartiniere ◽  
Cindy S. Dieringer ◽  
Etsuko Kita ◽  
George W. Lucier
Keyword(s):  
Enzyme Activity ◽  
Adult Male ◽  
Sexual Differentiation ◽  
Reproductive Tract ◽  
Testosterone Propionate ◽  
Hormone Treatment ◽  
Male Rats ◽  
Ventral Prostate ◽  
Uridine Diphosphate ◽  
Neonatal Administration

The hepatic microsomal enzyme UDP-glucuronyltransferase undergoes a complex developmental pattern in which enzyme activity is first detectable on the 18th day of gestation in rats. Prepubertal activities are similar for males and females. However, postpubertal sexual differentiation of enzyme activity occurs in which male activities are twice those of females. Neonatal administration of testosterone propionate or diethylstilboestrol to intact animals resulted in lowered UDP-glucuronyltransferase activity in liver microsomal fractions of adult male rats, whereas no changes were observed in the adult females and prepubertal male and female animals. Neonatal administration of testosterone propionate and diethylstilboestrol adversely affected male reproductive-tract development as evidenced by decreased weights of testes, seminal vesicles and ventral prostate. Diethylstilboestrol also markedly decreased spermatogenesis. Hypophysectomy of adult male rats resulted in negative modulation of microsomal UDP-glucuronyltransferase and prevented the sexual differentiation of enzyme activity. In contrast hypophysectomy had no effect on female UDP-glucuronyltransferase activity. A pituitary transplant under the kidney capsule was not capable of reversing the enzyme effects of hypophysectomy, therefore suggesting that the male pituitary factor(s) responsible for positive modulation of UDP-glucuronyltransferase might be under hypothalamic control in the form of a releasing factor. Neonatal testosterone propionate and diethylstilboestrol administration apparently interfered with the normal sequence of postpubertal UDP-glucuronyltransferase sexual differentiation.

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