Steroid Δ4-Reductases of Pig Liver: Partial Purification of Testosterone 5β-Reductase

1971 ◽  
Vol 49 (3) ◽  
pp. 385-392 ◽  
Author(s):  
J. C. Nduaguba ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Soluble Fraction ◽  
Reductase Activity ◽  
Maximum Velocity ◽  
Supernatant Fraction ◽  
Partial Purification ◽  
Pig Liver ◽  
Total Enzyme Activity ◽  
Distribution Studies ◽  
The Stability

Intracellular distribution studies of steroid Δ4-reductase activity in female pig liver were done using testosterone as substrate. About 60% of the total enzyme activity was found in the 100 000 × g soluble fraction. Labelled 17β-hydroxy-5β-androstan-3-one but not 17β-hydroxy-5α-androstan-3-one was isolated from the incubation of 1,2-3H-testosterone with the 100 000 × g supernatant fraction, indicating the presence of 5β-reductase activity. 5β-Reduction may play an important role in the inactivation of some Δ4-3-ketosteroids in the pig liver.Evidence that 5β-reductases differing in substrate specificity are present in the soluble fraction includes (a) variation in the ratios of enzyme activities for several Δ4-3-ketosteroids in different (NH4)2SO4 fractions obtained from the 100 000 × g soluble fraction, (b) kinetic data showing that the maximum velocity for an equimolar mixture of testosterone and hydrocortisone is the sum of the maximum velocities for the substrates when used singly, and (c) separation of the enzyme activity specific for testosterone from that specific for hydrocortisone by use of Sephadex G-100 and hydroxylapatite chromatography.Utilizing (NH4)2SO4 precipitation and chromatography on Sephadex G-100 and hydroxylapatite, a 105-fold purification of testosterone Δ4-5β-reductase from the 100 000 × g supernatant fraction has been attained. The presence of 5 mM β-mercaptoethanolamine increased the stability of the enzyme.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

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 Conditions that result in the mobilization and influx of Ca2+ into rat hepatocytes induce the rapid loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity that is not reversed by phosphatase treatment

1990 ◽  
Vol 269 (2) ◽  
pp. 373-379 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Enzyme Activity ◽  
Okadaic Acid ◽  
Reductase Activity ◽  
Total Activity ◽  
Ionophore A23187 ◽  
Hmg Coa Reductase ◽  
Pronounced Increase ◽  
Phosphorylation State ◽  
Total Enzyme Activity ◽  
Coa Reductase

We investigated the effects of conditions that induce Ca2+ mobilization from intracellular stores and Ca2+ influx into hepatocytes on the expressed and total (fully dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Vasopressin and phenylephrine when added alone had small or negligible effects on the phosphorylation state of the enzyme, as judged from the expressed/total activity ratio. However, when added in combination with glucagon, they elicited appreciable increases in the phosphorylation of the enzyme. Glucagon on its own had no effect either on phosphorylation state or on total HMG-CoA reductase activity during 40 min of incubation. Under conditions of sustained Ca2+ influx (i.e. vasopressin or phenylephrine plus glucagon), there was a marked loss of total HMG-CoA reductase activity. This effect was more pronounced when vasopressin was used; 50% of the enzyme activity was lost within 40 min. The involvement of Ca2+ in these effects was verified directly by the use of ionophore A23187. Its addition to hepatocytes resulted both in a very pronounced increase in the phosphorylation state of the enzyme and in the loss of 50% of the total activity within 30 min. There was no correlation between the ability of any set of conditions to increase the phosphorylation of the enzyme and the subsequent loss of total HMG-CoA reductase activity. The latter parameter appeared to be directly related, however, to the maintenance of prolonged Ca2+ influx, as indicated by the continued activation of glycogen phosphorylase, measured in the same cells. The lack of a causal relationship between increased phosphorylation and loss of total activity was demonstrated directly by studies in which okadaic acid was used to induce phosphorylation of HMG-CoA reductase in hepatocytes by inhibition of phosphatase 1 and 2A activities. This was not accompanied by any loss of total enzyme activity. Neither did okadaic acid enhance the loss of reductase induced by A23187 when the two agents were added together. It is concluded that altered Ca2+ fluxes in hepatocytes in vivo, under conditions of acute or chronic stress (such as may be associated with trauma or diabetes respectively), may be involved in the regulation of the expression of HMG-CoA reductase activity through alteration of enzyme concentration in the liver.

scholarly journals The biochemical basis for the conjugation of bile acids with either glycine or taurine

1978 ◽  
Vol 174 (2) ◽  
pp. 621-626 ◽  
Author(s):  
Donald A. Vessey
Keyword(s):  
Guinea Pig ◽  
Bile Acids ◽  
Soluble Fraction ◽  
Rabbit Liver ◽  
Supernatant Fraction ◽  
Pig Liver ◽  
Biochemical Basis ◽  
High Affinity ◽  
Guinea Pig Liver ◽  
Soluble Fractions

All animals, except for the placental mammals, conjugate their bile acids exclusively with taurine. However, in certain of the placental mammals, glycine conjugates are also found. The basis for the appearance of glycine conjugation among the placental mammals was investigated. The reaction of choloyl-CoA with glycine and taurine, as catalysed by the soluble fraction from guinea-pig liver, had a high affinity for taurine and a poor affinity for glycine. The predominant synthesis of glycine conjugates in the guinea pig can be related to the fact that guinea-pig liver contains an unusually low concentration of taurine and a high concentration of glycine. Rabbits make exclusively glycine conjugates and their livers also contain low concentrations of taurine. However, the biochemical basis for their glycine conjugation is more straightforward than in the guinea pig in that the soluble fraction from rabbit liver has a high affinity for glycine and a poor affinity for taurine. Alternative-substrate-inhibition studies with glycine and taurine in soluble fractions from guinea-pig and rabbit liver revealed that glycine and taurine were mutually inhibitory. This suggests that there is only one enzyme for glycine and taurine conjugation in these tissues. The soluble fractions from bovine liver and human liver also made both glycine and taurine conjugates and evidence is presented that suggests that there is only one enzyme in these tissues too. Even the rat, which excretes mostly taurine conjugates, could make both glycine and taurine conjugates in vitro. However, in contrast with all of the placental mammals studied, the supernatant fraction from liver of the chicken, and other non-mammals, could not make glycine conjugates even in the presence of very high concentrations of glycine.

scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

scholarly journals The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

2022 ◽  
Vol 2022 ◽  
pp. 1-7
Author(s):  
Yandri Yandri ◽  
Ezra Rheinsky Tiarsa ◽  
Tati Suhartati ◽  
Heri Satria ◽  
Bambang Irawan ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Half Life ◽  
Thermal Inactivation ◽  
Immobilized Enzyme ◽  
Residual Activity ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Optimum Temperature ◽  
The Stability

The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, V max of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, V max of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

scholarly journals Dipeptidase activity in the small intestinal mucosa during pregnancy and lactation in the rat

1975 ◽  
Vol 33 (1) ◽  
pp. 1-9 ◽  
Author(s):  
B. A. Rolls
Keyword(s):  
Enzyme Activity ◽  
Intestinal Mucosa ◽  
Soluble Fraction ◽  
Supernatant Fraction ◽  
Small Intestinal Mucosa ◽  
Pregnancy And Lactation ◽  
Specific Activities ◽  
Small Intestinal ◽  
Hormonal Stimulus ◽  
Dipeptidase Activity

1. Rats were mated and at weekly intervals during pregnancy and lactation, and after weaning, the dipeptidase activity in the supernatant fraction from small intestinal mucosa extracts was determined for two dipeptides: glycyl–L-leucine dipeptidase (EC 3.4.3.2) and L-alanyl–L-glutamic acid dipeptidase.2. Dipeptidase activity is found mainly in the soluble (supernantant) fraction of the mucosa homogenate.3. Compared with those values obtained for unmated controls, the food consumption of the animals and the nitrogen content, total and specific activities of the dipeptidases (per unit quantity of N) in the soluble fraction of the small intestinal mucosa extracts increased slightly during pregnancy and markedly during lactation. After the pups were weaned, values for all these measurements fell rapidly.4. Whether the increases found in enzyme activity were simply a response to increased food intake or were the result of hormonal stimulus is discussed.

Characterization of acetylcholinesterase in rabbit intrapulmonary arteries

1994 ◽  
Vol 267 (6) ◽  
pp. L745-L752 ◽  
Author(s):  
R. J. Altiere ◽  
D. C. Travis ◽  
D. C. Thompson
Keyword(s):  
Enzyme Activity ◽  
Blood Vessels ◽  
Cholinesterase Activity ◽  
Maximum Velocity ◽  
Pulmonary Vasculature ◽  
Cholinesterase Inhibition ◽  
Moderate Amount ◽  
Peripheral Tissues ◽  
Total Enzyme Activity ◽  
Cholinesterase Activities

Acetylcholine (ACh) acts on the pulmonary vasculature to evoke vasodilation and, in some species, vasoconstriction. The actions of ACh are terminated by its rapid hydrolysis by cholinesterases. Aside from histochemical localization studies, there is little information on cholinesterase enzymes in pulmonary blood vessels. The present study addresses the hypothesis that pulmonary blood vessels contain sufficient cholinesterase activity to regulate the action of ACh in these tissues. Accordingly, studies were undertaken to characterize and quantify cholinesterase activities in pulmonary arteries and veins, quantify inhibition of enzyme activity, and investigate functional physiological consequences of cholinesterase inhibition. Cholinesterase activities in aorta and trachea also were examined for comparison. Kinetic studies showed that the lobar pulmonary arterial enzyme has a Michaelis constant of 55.3 +/- 17.0 microM and a maximum velocity of 8.6 +/- 2.7 nmol/min/mg protein similar to cholinesterases found in other peripheral tissues. Studies with selective inhibitors revealed that > 98% of total enzyme activity was attributable to acetylcholinesterase. Similar levels of enzyme activity were found in homogenates of lobar branch intrapulmonary arteries, intrapulmonary veins, and aorta. The majority of enzyme activity was localized to the membrane fraction, although a moderate amount was found in the cytosol. Studies in intact intrapulmonary lobar arteries showed that these vessels had cholinesterase activity comparable with that found in intact trachealis muscle and that neostigmine (10 nM to 10 microM) caused concentration-dependent inhibition of enzyme activity. In isolated intrapulmonary lobar arteries, functional studies showed that 1 and 10 microM neostigmine significantly potentiated ACh-induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Resolution of the phosphoinositide-specific phospholipase C isolated from porcine lymphocytes into multiple species. Partial purification of two isoenzymes

1987 ◽  
Vol 244 (3) ◽  
pp. 639-645 ◽  
Author(s):  
H R Carter ◽  
A D Smith
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Soluble Form ◽  
Partial Purification ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Mesenteric Lymph ◽  
Multiple Species

Phospholipase C isolated from porcine mesenteric lymph node lymphocytes was distributed between the soluble and particulate fractions. Enzyme activity was found predominantly in the soluble fraction with optimal activity at pH 5.5. Gel filtration chromatography of the soluble phospholipase C revealed that it was composed of multiple species of enzyme activity. The activity associated with the particulate fraction had optimal activity at pH 7.0, as also did one of the species of soluble phospholipase C. Cellulose phosphate chromatography resolved the major soluble form into two species designated PLC-A and PLC-B. Both phenyl-Sepharose chromatography and hydroxyapatite chromatography purified these species still further. PLC-A and PLC-B demonstrated similar activities against phosphatidylinositol with a pH optimum near 5.5. The phospholipase C activities were abolished against this substrate by the addition of 1 mM-EDTA. When assayed in the presence of Ca2+-EDTA buffers providing a range of Ca2+ free concentrations, both enzymes exhibited optimal activity near 10(-3) M free Ca2+, but PLC-B was inhibited above this concentration more than PLC-A. PLC-B exhibited markedly lower activity against phosphatidylinositol 4,5-bisphosphate, suspended as liposomes of the pure phospholipid, than did PLC-A.

Pengaruh Penambahan Molases Pada Kulit Pisang Kapendis Untuk Media Produksi Xilanase B.Stearothermophillus DSM 22

2019 ◽  
Vol 15 (3) ◽  
Author(s):  
Trismillah
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Banana Peel ◽  
Partial Purification ◽  
Temperature Conditions ◽  
Cavendish Banana ◽  
Working Volume ◽  
Initial Ph ◽  
Xylanase Enzyme

Cavendish banana peel can be used as a substitute for the expensive xylan, while molasses than as a source of carbon as well as nitrogen, minerals and nutrients needed for the growth of microbes that can produce the enzyme. Xylanase produced from Bacillus stearothermopillus DSM 22, using media cavendish banana peels with the addition of molasses 1%, 2%, and 3%. Fermentation is done in a shaker incubator at 550C temperature conditions, initial pH 8, and 250 rpm agitation. The result showed the highest enzyme activity of 4,14 ± 0,16 U/mL min., on the addition 2% molasses after 24 hours. Further fermentation carried out in the fermenter working volume of 3.5 liters, with the condition of temperature 550C, pH 8, aeration 1 vvm, agitation 250 rpm, the highest spesific enzyme of activity of 51,62 ± 0,16 U/mg after 24 hours. Partial purification of xylanase enzyme fermentation is done with the results of microfiltration, ultrafiltration, ammonium sulfate (0-80%) and dialysis. There is an increase in the purity of the enzyme at each stage of purification, the highest purity on dialysis 3.23 times of crude enzymes.Kulit buah pisang kapendis dapat digunakan sebagai pengganti xilan yang harganya mahal, sementara molases selain sebagai sumber karbon serta nitrogen, mineral dan nutrisi dibutuhkan untuk pertumbuhan mikroba yang dapat menghasilkan enzim. Xilanase yang dihasilkan dari Bacillus stearothermopillus DSM 22, menggunakan media kulit pisang kapendis dengan penambahan molase 1%, 2%, dan 3%. Fermentasi dilakukan dalam shaker inkubator pada temperatur 550C, pH awal 8, dan agitasi 250 rpm. Hasilnya menunjukkan aktivitas enzim tertinggi 4,14 ± 0,16 U/mL min., pada penambahan 2% molases setelah 24 jam. Selanjutnya fermentasi dilakukan di dalam fermentor, volume kerja dari 3,5 liter, dengan kondisi temperatur 550C, pH 8, aeration 1 vvm, agitasi 250 rpm, aktivitas spesifik tertinggi 51,62 ± 0,16 U/mg setelah 24 jam. Pemurnian parsial fermentasi enzim xilanase dilakukan dengan hasil mikrofiltrasi, ultrafiltrasi, amonium sulfat (0-80%) dan dialisis. Ada peningkatan kemurnian enzim pada setiap tahap pemurnian, kemurnian tertinggi pada dialisis 3,23 kali dari enzim kasar.Keywords: Xylanase, B. stearothermophillus DSM 22, Cavendish banana peel, molasses, enzyme activity

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