Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg²⁺, whereas inhibited strongly by Cu²⁺ and Zn²⁺. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.     

Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe⁺³ and Cu⁺². Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro.Incorporation of [¹⁴C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom.The venom demonstrated activity against carbobenzoxy-l-tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10^?3m levels of EDTA and EGTA but not by 2.5 × 10^?4mo-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Alkalinity within the midgut of mosquito larvae with alkaline-active digestive enzymes.

Digestive enzyme pH optima were determined in vitro for the larvae of three mosquito spp., Culex pipiens, Aedes aegypti, and Anopheles stephensi. All had optimal amylase activity at about pH 8 and optimal protease activity over a broad range between pH 10 and 12. pH within the digestive tract of intact live larvae was determined from the colours of indicator dyes ingested with kaolin and visible in the gut through the transparent or translucent body tissues. In all three spp. the contents of the anterior midgut were held at a pH just exceeding 10, with an abrupt fall to about pH 7·5 in the posterior third midgut.     

Purification and properties of three kinds of α-hydroxysteriod dehydrogenases from Brevibacterium fuscum DC33

The cell-free extract of Brevibacterium fuscum DC33 contained three kinds of hydroxysteriod dehydrogenase (3a-, 7a-, and 12a-hydroxysteriod dehydrogenases). 7a-Hydroxysteroid dehydrogenase (EC 1.1.1.59) was purified to electrophoretical homogeneity by ion exchange chromatography, affinity chromatography, and preparative electrophoresis. Its molecular weight was 104, 000 and the enzyme was composed of four identical subunits. The enzyme had an optimum pH of 5.3 for dehydrocholic acid reduction, and around 10 for cholic acid oxidation. It was stable in a pH range of 5.7 to 10.5 at 5°C overnight. The enzyme was most active at 25° to 30°C. The activity was not affected by incubation at 30°C for 30 min, but it was lost at 40°C for 30 min. Withe the assumption of two-substrate kinetics, we calculated various kinetic constants for dehydrocholic acid, 7, 12-diketolithocholic acid, 12-ketochenodeoxycholic acid, and 3, 12-diketolithocholic acid (for the structure of bile acids, see Table 2) together with NAD⁺ or NADH. The enzyme was active only toward hydroxysteroids with a 7a-hydroxyl group. The production of 7-ketochenodeoxycholic acid from cholic acid and of 3, 12-diketolithocholic acid from dehydrocholic acid by the purified 7a-hydroxysteroid dehydrogenase was confirmed by thin-layer chromatography.12a-Hydroxysteroid dehydrogenase was purified by a similar method. It was active toward hydroxysteroids with a 12a-hydroxyl group.3a-Hydroxysteroid dehydrogenase was purified by preparative electrophoresis. It was active toward hydroxysteroids with a 3a-hydroxyl group.     

Invertases of pollen of Haemanthus albiflos

Soluble and insoluble invertase occurs in dormant pollen of Haemanthus albiflos, with pH optima of 5·7 and 5·5 respectively. At their pH optima the activity of the soluble enzyme is 3·5-fold higher. After 2 hr germination the pH optimum of the insoluble invertase is increased to 6·0 and the activity is increased 2-fold while the activity of the soluble invertase is decreased by 26%.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

Biosynthesis and genetic control of isovitexin 7-O-xyloside in the petals of melandrium album

An enzyme was detected in petal extracts of Melandrium album which catalysed the transfer of the xylose moiety of UDP-xylose to the 7-hydroxyl group of isovitexin. Genetical analysis revealed that the presence of the dominant allele g^x was necessary for enzymic activity. This activity was independent of the residual genetic background. Xylosyltransferase activity is also present in extracts of g^Gg^x plants, in which the product of the enzyme is not detectable. Maximal activity was found between pH 7·0 and 7·5; MnCl₂ inhibited this transfer. The enzyme had an ‘apparent K_m' value of 1·0 mM for UDP-xylose and of O·04 mM for isovitexin.     

Lipoxygenase in Vicia faba minor

Lipoxygenase activity was demonstrated in partially purified preparations from small faba beans. The enzyme was shown to possess a pH optimum of 6·5 and was inactivated by exposure to 70° for 15 min. The K_m value for linoleic acid was calculated to be 0·57 mM. Ammonium sulphate fractionation yielded two highly active preparations, which were both active towards linoleic and linolenic acids. Neither fraction was inhibited by either cyanide or p-chloromercuribenzoate. The two fractions showed markedly differing responses to calcium ions, suggesting the presence of two lipoxygenases in faba beans. Activation of the enzyme by calcium ions was eliminated by the addition of EDTA.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Effect of pH on the modulation of rat osseous plate alkaline phosphatase by metal ions.

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Purification and characterization of secreted acid phosphatase under phosphate-deficient condition inPholiota nameko

Activity of acid phosphatase secreted by mycelia ofPholiota nameko on cultivation for 30d in Pi-depleted medium was 88-fold higher than the corresponding activity in the Pi-supplied medium. One isozyme of the secreted acid phosphatases was purified from the culture filtrate of Pi-depleted medium by ammonium sulfate fractionation and cation exchange chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed change chromatography. The purified enzyme was homogeneous on electrophoresis. Gel filtration analysis showed that the native molecule had a molecular weight of 117,000. The molecular weight on gel electrophoresis with SDS was 52,000, indicating that the native form of the enzyme was a homodimer. The optimum pH and temperature of the enzyme were, 5.5 and 45°C, respectively, and the isoelectric point of the enzyme was pH 6.9. Adsorption on Con A-Sepharose and periodic-Schiff stain suggested that the enzyme is a glycoprotein. The enzyme hydrolyzed a wide variety of phosphate esters, nucleoside phosphates, sugar phosphates, and phosphorylated amino acids. Cu²⁺, Fe²⁺, Hg²⁺, iodoacetate, molybdate, tartaric acid, and SDS inhibited the enzyme activity. Fe³⁺ (1 mM), Triton X-100, methanol, and ethanol activated it. Fifteen residues of the N-terminal amino acid sequence were determined.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

SOME PHYSICAL PROPERTIES OF BOVINE ADRENAL MEDULLARY DOPAMINE β-HYDROXYLASE

(1) Dopamine, β-hydroxylase (EC 1.14.2.1) was purified from bovine adrenal medullae according to the method of Foldes , Jeffrey , Preston and Austin (1972). (2) The kinetics, pH optimum and the effect of Cu²⁺ ions on the purified enzyme were found to resemble those of the enzyme isolated by more involved procedures. (3) The sedimentation coefficient (s₂₀) of the homogeneous enzyme in 10 mM-phosphate buffer, pH 7·2, containing 0·1 M-NaCI was found to be 10·24 ± 0·12 (S.E.M. of 10 determinations). (4) The effect of pH on the mol. wt. of the enzyme was investigated and no large deviation was found from the native mol. wt. of 290,000 in the pH range 3·9 to 11·1. (5) The amino acid analysis of dopamine β-hydroxylase is presented, and is contrasted to that of chromogranin A purified from the same chromaffin granule lysate. (6) Treatment with either 8 M-urea or 0·1% (w/v) sodium dodecyl sulphate was found to dissociate the enzyme into three similar, non-active subunits, each of mol. wt. of the order of 100,000.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

Haemolymph lipase activity in the American cockroach, Periplaneta americana

The haemolymph of the cockroach, Periplaneta americana, has been shown to possess pronounced lipolytic activity. The enzyme may be considered a ‘true’ lipase because of its ability to hydrolyse long chain fatty acid glycerides. The enzyme exhibits dual optima at pH 4·9 and 7·0 and is inhibited by fluoride ion. Lipase activity in the intact cockroach is unaffected by the corpus cardiacum hypolipemic factor; however, prolonged periods of starvation result in a significant decline in enzyme activity.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.