scholarly journals Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 119 (4) ◽  
pp. 635-642 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Enzyme Preparation ◽  
Reaction Rate ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Freezing And Thawing ◽  
Cytosol Fraction ◽  
Tris Maleate ◽  
Conjugating Enzyme

An enzyme that conjugates the 17β-hydroxyl group of testosterone was found in the cytosol fraction of human liver. The same enzyme preparation also conjugates the 16α-hydroxyl group of oestriol. The enzymic activity could not be sedimented by centrifuging the cytosol fraction at 158000gav. for 120min. The testosterone-conjugating as well as the oestriol-conjugating activities were found in the precipitate obtained after 30% saturation of the cytosol fraction with ammonium sulphate. Filtration of the precipitate through Sephadex G-200 enriched the testosterone-conjugating enzyme 50-fold and the oestriol-conjugating enzyme 100-fold. No separation of the two activities was achieved. With labelled testosterone the product of the reaction, testosterone 17β-glucuronide, was identified by paper chromatography and by crystallization to constant specific radioactivity. Testosterone 17β-glucuronyltransferase was active between pH7.0 and 8.6 in tris–HCl and tris–maleate buffers. The apparent Km values for testosterone and UDP-glucuronic acid were 6.4 and 25μm respectively. The enzyme was active between 37 and 45°C; the activation energy was calculated to be 5kcal/mol. Oestriol did not influence the glucuronidation of testosterone. Controlled heating as well as alternate freezing and thawing of the purified enzyme preparation led to an inactivation of both testosterone-conjugating and oestriol-conjugating activities at similar rates. Testosterone and oestriol, when incubated together, gave a reaction rate that was approximately equal to the sum of the rates when the two substrates were incubated separately. The present findings suggest that testosterone and oestriol are conjugated by two separate enzymes.

scholarly journals Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 118 (4) ◽  
pp. 625-634 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Glucuronic Acid ◽  
Specific Radioactivity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Partial Purification ◽  
Cytosol Fraction ◽  
Enzyme Substrate ◽  
Kinetics Of

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000gav. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu2+, Zn2+ and Hg2+ inhibited, whereas Mg2+, Mn2+ and Fe2+ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.

UDP-Glucose: Anthocyanidin/FIavonol 3-O-Glucosyltransferase in Enzyme Preparation from Flower Extracts of Genetically Defined Lines of Matthiola incana R. Br.

1986 ◽  
Vol 41 (7-8) ◽  
pp. 699-706 ◽  
Author(s):  
M. Teusch ◽  
G. Forkmann ◽  
W. Seyffert
Keyword(s):  
Enzyme Activity ◽  
Structural Gene ◽  
Enzyme Preparation ◽  
Reaction Rate ◽  
Flower Colour ◽  
Hydroxyl Group ◽  
Anthocyanin Synthesis ◽  
Ph Optimum ◽  
Matthiola Incana ◽  
Highly Active

Abstract In flower extracts of Matthiola incana an enzyme catalyzing the transfer of glucose from UDP- glucose to the hydroxyl group at 3-position of anthocyanidins and flavonols was demonstrated. The pH-optimum of this reaction is at pH 8.5 for pelargonidin and pH 9.5 for quercetin as substrate. The reaction is inhibited by both substrates above 10 nmol per assay. The enzyme is highly active, within 30 sec 3 nmol of 3-glucosides were formed. At 30 °C the enzyme is stable for hours and at -20 °C months. Besides UDP-glucose, TDP-glucose is a suitable glucosyl-donor, but with a reduced (70%) reaction rate. Enzyme activity is clearly inhibited by Fe2+ and Cu2+ ions, and by diethylpyrocarbonate. Acyanic or pale coloured mutants of several genes interfering with anthocyanin synthesis after dihydroflavonol formation show a more or less drastically reduced enzyme activity (5-40%). But none of these genes can be regarded as the structural gene for the 3-glucosyltransferase. The influence of these genes on enzyme activity and flower colour is dis­cussed.

scholarly journals Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1965 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
PJ Peterson ◽  
L Fowden
Keyword(s):  
Carboxylic Acid ◽  
Enzyme Preparation ◽  
Enzymic Activity ◽  
The Other ◽  
Asparagus Officinalis ◽  
Acid Activation ◽  
Phaseolus Aureus ◽  
Substrate Specificities ◽  
Imino Acids ◽  
Ammonium Sulphate Fractionation

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

THE KINETICS OF AMINO GROUP TRANSFER BY CORN RADICLE TRANSAMINASE

1957 ◽  
Vol 35 (12) ◽  
pp. 1289-1303 ◽  
Author(s):  
F. S. Cook
Keyword(s):  
Classical Theory ◽  
Spectrophotometric Method ◽  
Substrate Concentration ◽  
Enzyme Preparation ◽  
Reaction Rate ◽  
Amino Group ◽  
Group Transfer ◽  
Kinetics Of ◽  
Published Account

The kinetics of transamination are complicated by the presence of two substrates whose concentrations change appreciably during the course of the reaction. The only previously published account of the kinetics of this system deviates considerably from classical theory. Equations based on premises of Michaelis and Menten have been shown, however, to accommodate the data on reaction rate in relation to substrate concentration obtained with a corn radicle enzyme preparation by a spectrophotometric method.

Determination of bromsulphthalein-glutathione conjugating enzyme activity in needle biopsy specimens of human liver

1973 ◽  
Vol 47 (2) ◽  
pp. 133-137 ◽  
Author(s):  
D.V. Datta ◽  
Sarban Singh ◽  
A.K.S. Samanta ◽  
S. Saha ◽  
M. Mukherjee ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Needle Biopsy ◽  
Human Liver ◽  
Biopsy Specimens ◽  
Conjugating Enzyme

scholarly journals DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

1966 ◽  
Vol 30 (3) ◽  
pp. 519-530 ◽  
Author(s):  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Pellet Fraction ◽  
Light Region ◽  
Centrifuge Tube ◽  
In Vivo Labeling

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

IV. Cysteine proteinases

1970 ◽  
Vol 257 (813) ◽  
pp. 231-236 ◽  
Keyword(s):  
Oxidation State ◽  
Proteolytic Enzyme ◽  
Enzyme Preparation ◽  
Chemical Nature ◽  
Enzymic Activity ◽  
Active Enzyme ◽  
Tropical Tree ◽  
Cysteine Proteinases ◽  
Sulphydryl Group ◽  
Sh Group

Papain (EC 3.4.4.10) is a proteolytic enzyme which is isolated from the Papaya, a common tropical tree. It is a sulphydryl enzyme and its SH group is required for enzymic activity. Papain as usually prepared (Kimmel & Smith 1954) contains only a small portion of active molecules. The majority of the molecules are inactive because their sulphydryl group is blocked. Part of the blocking is caused by disulphide formation with cysteine (Sluyterman 1967). This disulphide can be reduced by an excess of cysteine resulting in an active enzyme preparation. The free SH content never reaches 100% and is often not more than about 50% , so that we must distinguish between papain molecules with a reversibly and an irreversibly blocked SH group. The chemical nature of the irreversible blocking is not yet known. It might well be due to a higher oxidation state of the sulphur which cannot be reduced by an excess of cysteine (Glazer & Smith 1965).

Studies on Partially Purified Pig Liver Steroid Δ4-5β-Reductase Activity

1973 ◽  
Vol 51 (12) ◽  
pp. 1661-1668 ◽  
Author(s):  
Edward J. Van Doorn ◽  
John C. Nduaguba ◽  
Albert F. Clark
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Reductase Activity ◽  
Enzymatic Reaction ◽  
Ph Optimum ◽  
Pig Liver ◽  
Liver Cytosol ◽  
End Products ◽  
Tris Maleate

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

scholarly journals Characterization of human liver α-D-mannosidase purified by affinity chromatography

1976 ◽  
Vol 153 (3) ◽  
pp. 579-587 ◽  
Author(s):  
N C Phillips ◽  
D Robinson ◽  
B G Winchester
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Human Liver ◽  
Early Stage ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Lysosomal Storage ◽  
Sepharose 4B

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

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