Extracellular alkaline phosphatase from alveolar secretions of patients with pulmonary alveolar proteinosis

1981 ◽  
Vol 59 (4) ◽  
pp. 290-300 ◽  
Author(s):  
Denis Nadeau ◽  
Mark J. Reasor ◽  
Gary E. R. Hook
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Pulmonary Alveolar Proteinosis ◽  
Extracellular Enzyme ◽  
Soluble Form ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
High Molecular Weight Complex ◽  
Heat Stable ◽  
Alveolar Proteinosis

Alkaline phosphatases in alveolar secretions from the lungs of patients with pulmonary alveolar proteinosis have been studied. A soluble form of alkaline phosphatase was isolated from the secretions and characterized. The extracellular enzyme had a pH optimum at 9.95; was stimulated by Mg2+, Ni2+, and Mn2+; was inhibited by Zn2+, Be2+, Cu2+, and low concentrations (8 mM) of L-homoarginine and imidazole; and was heat-stable at 55 °C. The soluble phosphatase existed primarily as a high molecular weight complex (excluded from Sepharose 4B) and could be dispersed into low molecular weight forms (205 000 – 285 000) by treatment with n-butanol. Following butanol treatment, the thermostability of the enzyme was markedly decreased but the kinetic properties such as the Km values, activation energies, and responses to various inhibitors were unchanged.The alkaline phosphatase may originate from unusual type 2 cells present in the alveoli of patients with pulmonary alveolar proteinosis.

scholarly journals Purification and properties of molecular-weight variants of human placental alkaline phosphatase

1968 ◽  
Vol 108 (5) ◽  
pp. 779-792 ◽  
Author(s):  
Nimai K. Ghosh ◽  
William H. Fishman
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Large Scale ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Exchange Chromatography ◽  
Placental Alkaline Phosphatase ◽  
Ph Optimum ◽  
Starch Gel

1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8·6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4·2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.

Partial characterization of the extracellular carboxymethylcellulase activity produced by the rumen bacterium Bacteroides succinogenes

1983 ◽  
Vol 29 (5) ◽  
pp. 504-517 ◽  
Author(s):  
D. Groleau ◽  
C. W. Forsberg
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Culture Fluid ◽  
Extracellular Enzyme ◽  
Rumen Bacterium ◽  
Ph Optimum ◽  
Complex Protein ◽  
Culture Supernate ◽  
Triton X

In cultures of Bacteroides succinogenes, in which cellulose was the source of carbohydrate, from 70 to 80% of the carboxymethylcellulase (CMCase) activity was present in the culture fluid. The crude extracellular enzyme readily hydrolyzed acid-swollen cellulose with the production of glucose and cellobiose. Of this extracellular CMCase, 50–62% was associated with sedimentable membrane fragments, 9–13% with nonsedimentable material with a molecular weight greater than 4 × 106, and 28–38% with molecules having a molecular weight of approximately 45 000. Polyacrylamide gel electrophoresis (PAGE), in the presence of sodium dodecyl sulfate, revealed that both the nonsedimentable and the sedimentable fraction had complex protein compositions. The nonsedimentable and sedimentable CMCase fractions, after treatment with Triton X-100, were subjected to PAGE in the presence of 0.2% (w/v) Triton X-100. The results indicated the presence of fast- and slow-migrating CMCases in the former, and of a slow-migrating CMCase in the latter. An apparently uncharged CMCase, which probably corresponded to the slow-migrating component by PAGE, was partially purified from the concentrated culture supernate by solubilization in Triton X-100 and chromatography on DEAE–Sepharose, CM–Sepharose, and Phenyl–Sepharose. The partially purified CMCase had a pH optimum of 5.6–6.6 and a temperature optimum of 50 °C.

scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

Purification and Properties of Phosphoenolpyruvate Carboxykinase from C4 Plants

1986 ◽  
Vol 13 (5) ◽  
pp. 577 ◽  
Author(s):  
JN Burnell
Keyword(s):  
Molecular Weight ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Kinetic Properties ◽  
Panicum Maximum ◽  
Ph Optimum ◽  
Wide Ph Range ◽  
Pep Carboxykinase ◽  
Nucleotide Specificity ◽  
Fructose 1,6 Bisphosphate

Phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.49) from the leaves of Urochloa panicoides, Chloris gayana and Panicum maximum has been purified to homogeneity and its properties determined. The enzyme from all three PEP carboxykinase-type C4 plants have similar physical and kinetic properties. The native enzyme has a molecular weight of 380 000 and its monomeric molecular weight is about 64 000, suggesting the enzyme is hexameric. It is active over a wide pH range and has a pH optimum between 7.4 and 8.2, has a wide nucleotide specificity, has an absolute requirement for Mn2+ and is stimulated by C-. The enzyme is inhibited by 3-phosphoglyceric acid, fructose 6-phosphate and fructose 1,6-bisphosphate; the mechanism of inhibition is discussed. The purified PEP carboxykinase is unable to catalyse the conversion of oxaloacetate to pyruvate, nor does it possess pyruvate kinase activity. These findings are discussed in relation to the C4 photosynthetic pathway operating in PEP carboxykinase-type C4 plants.

scholarly journals Properties of inorganic pyrophosphatase of pig scapula cartilage

1975 ◽  
Vol 147 (1) ◽  
pp. 111-118 ◽  
Author(s):  
R Felix ◽  
H Fleisch
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Ammonium Molybdate ◽  
Inorganic Pyrophosphatase ◽  
Ph Optimum ◽  
Km Value ◽  
Calcification Process

The properties of a highly purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) from pig scapula cartilage were studied. The enzyme had a molecular weight of 66 000 and a pH optimum of 7-8. It was markedly activated by magnesium, but not, or only to a much smaller degree, by other metal ions. PP1 was the only substrate found and had a Km value of 11 muM. The enzyme was not inhibited by phosphate and other inhibitors of alkaline phosphatase such as CN- minus, amino acids and theophylline; it was slightly inhibited by tartrate, formaldehyde and ammonium molybdate and strongly inhibited by F- minus, Ca2+ and other metal ions. The properties of the enzyme in the presence of concentrations of PP1 present in plasma (3.5 muM) were similar to those found at higher (2 mM) concentrations of PP1. The diphosphonates ethane-1-hydroxy-1,1-diphosphonate and dichloromethylenediphosphonate inhibited the enzyme in the presence of low PP1 concentrations. The characteristics of this enzyme are therefore similar to pyrophosphatases from other sources, such as from yeast and erythrocytes, and do not support a specific role of this enzyme in the calcification process.

scholarly journals Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen

1984 ◽  
Vol 217 (3) ◽  
pp. 675-683 ◽  
Author(s):  
D C Williams
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Kinetic Properties ◽  
Stable Form ◽  
Multiple Forms ◽  
Ph Optimum ◽  
Heat Stable ◽  
Decreasing Ph ◽  
Covalent Intermediate

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.

DEGRADATION OF THE POLYSACCHARIDE OF XANTHOMONAS PHASEOLI BY AN EXTRACELLULAR BACTERIAL ENZYME

1961 ◽  
Vol 7 (5) ◽  
pp. 815-825 ◽  
Author(s):  
S. M. Lesley
Keyword(s):  
Molecular Weight ◽  
Glucuronic Acid ◽  
Degradation Products ◽  
Extracellular Enzyme ◽  
Low Molecular Weight ◽  
Acid Moiety ◽  
Ph Optimum ◽  
Polysaccharide Degradation ◽  
Bacterial Enzyme ◽  
Stability Examination

A species of Bacillus isolated from soil was found to elaborate an inducible, extracellular enzyme capable of degrading the polysaccharide of Xanthomonas phaseoli to low-molecular-weight oligosaccharides. The enzyme was characterized with respect to pH optimum, ion requirement, and stability. Examination of the polysaccharide degradation products revealed that the glucuronic acid moiety of the polysaccharide was altered to a Δ4,5 unsaturated form as a result of enzyme action.

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