scholarly journals Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

1972 ◽  
Vol 128 (4) ◽  
pp. 817-831 ◽  
Author(s):  
A. Anne Malcolm ◽  
M. G. Shepherd
Keyword(s):  
Molecular Weight ◽  
Thermal Inactivation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Thermophilic Fungus ◽  
Sucrose Density Gradient Centrifugation ◽  
Coenzyme Specificity ◽  
Heat Stable ◽  
Purification And Properties ◽  
Glucose 6 Phosphate Dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000±10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP+- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP+, protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The Km values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3×10−5m-NADP+ and 1.6×10−4m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2×10−5m-NADP+ and 2.5×10−4m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP+ and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme–NADP+–6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ·mol−1 (9.6 and 9.9kcal·mol−1) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5×10−6m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

Purification and properties of ι-carrageenase from a marine bacterium

1984 ◽  
Vol 30 (12) ◽  
pp. 1500-1506 ◽  
Author(s):  
C. W. Greer ◽  
W. Yaphe
Keyword(s):  
Marine Bacterium ◽  
Culture Medium ◽  
Gradient Centrifugation ◽  
Hydrophobic Interaction Chromatography ◽  
Apparent Molecular Weight ◽  
Sucrose Density Gradient ◽  
Resonance Spectroscopy ◽  
Sucrose Density Gradient Centrifugation ◽  
Purification And Properties ◽  
Wall Components

An ι-carrageenase has been purified from the cell-free culture medium of a marine bacterium grown in ι-carrageenan. The enzyme hydrolyzes the β1 → 4 linkages in ι-carrageenan; the major end products, as identified by 13C nuclear magnetic resonance spectroscopy, are ι-neocarratetraose sulfate and ι-neocarrahexaose sulfate. The enzyme was purified by fractionation on Sephacryl S-200 in 2.0 M NaCl and hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. The purified enzyme had an apparent molecular weight of 57 000, and optimum activity was expressed at pH 8.0, 40 °C, in 0.1 M Na+. The enzyme was stable for at least 6 months at 4 °C in 1.0 M NaCl. Removal of the salt, freezing, or lyophilization destroyed enzyme activity. Membrane fractions, prepared by discontinuous sucrose density gradient centrifugation, also exhibited ι-carrageenase activity. Properties of ι-carrageenase suggest an association with cell wall components.

scholarly journals Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).

1988 ◽  
Vol 107 (5) ◽  
pp. 1767-1776 ◽  
Author(s):  
M D Neely ◽  
K Boekelheide
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sertoli Cell ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytoplasmic Dynein ◽  
Microtubule Assembly ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Stage ◽  
Structural And Functional Properties

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
Author(s):  
A. R. EASTMAN ◽  
A. M. NEVILLE
Keyword(s):  
Molecular Weight ◽  
Adrenal Glands ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Molecular Sizes ◽  
Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

scholarly journals Appearance of a methylated ribonucleic acid on illumination of Euglena gracillis cells grown in the dark

1971 ◽  
Vol 125 (2) ◽  
pp. 401-405 ◽  
Author(s):  
M. Perl
Keyword(s):  
Molecular Weight ◽  
Nucleic Acid ◽  
High Molecular Weight ◽  
Ribonucleic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Acid Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Exclusion Chromatography

Investigation of the methylation of nucleic acids by [Me-3H]methionine after illumination of Euglena cells grown in the dark has shown that a high-molecular-weight nucleic acid fraction undergoes methylation after exposure to light for 60–120min. This methylated nucleic acid fraction was isolated both by sucrose-density-gradient centrifugation and exclusion chromatography on Sephadex G-200. The fraction was shown to consist of a preformed RNA that is present in cells grown in the dark and which on illumination is transmethylated by methionine.

Purification and properties of mycoferritin from Mortierella alpina

1972 ◽  
Vol 18 (5) ◽  
pp. 619-622 ◽  
Author(s):  
R. F. Bozarth ◽  
A. Goenaga
Keyword(s):  
High Speed ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Potassium Phosphate ◽  
Sucrose Density Gradient ◽  
Mortierella Alpina ◽  
Protein Subunit ◽  
High Concentration ◽  
Sucrose Density Gradient Centrifugation ◽  
Purification And Properties

A culture of Mortierella alpina Peyroud isolated from soil and grown on Czapek-Dox medium was found to contain a high concentration of mycoferritin (MF). The MF was extracted from lyophilized mycelium by grinding with 0.1 M, pH 7.0, potassium-phosphate buffer and chloroform followed by differential centrifugation. Sucrose density-gradient centrifugation of the concentrated MF resulted in a brownish-yellow band in the region of 60–70 S. Following dialysis and concentration by high-speed centrifugation, the MF was compared to horse-spleen ferritin (HF). The ultraviolet (uv.) and infrared (i.r.) spectra of MF and HF were identical. Negatively stained preparations examined in the electron microscope showed particles of about 10 nm diameter. Sedimentation rates of S20,w = 66 for MF and S20,w = 56 for HF were obtained by analytical ultracentrifugation. The MF preparation contained 83% protein and 17% iron. The molecular weight of the protein subunit was determined by gel electrophoresis to be about 19 300 daltons.

A malate dehydrogenase of high molecular weight, from bean leaves

1968 ◽  
Vol 46 (5) ◽  
pp. 719-720 ◽  
Author(s):  
William Habig ◽  
David Racusen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Malate Dehydrogenase ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights ◽  
Young Leaves ◽  
Bean Leaves

Two forms of malate dehydrogenase (MDH) of widely differing molecular weight were found in primary leaves of Phaseolus vulgaris. Their molecular weights were estimated as 69 000 and 275 000 by sucrose density gradient centrifugation. The ratio of these two forms followed an orderly course in which the high molecular weight MDH increased from near zero in very young leaves to about 35% of the total MDH activity in leaves older than 2 weeks. Conditions which cause the high molecular weight MDH to dissociate to active normal molecular weight enzyme are discussed.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

scholarly journals Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L.

1968 ◽  
Vol 106 (3) ◽  
pp. 689-698 ◽  
Author(s):  
T. A. Dyer ◽  
Rachel M. Leech
Keyword(s):  
Molecular Weight ◽  
Nucleic Acids ◽  
Serum Albumin ◽  
Vicia Faba ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Low Molecular Weight ◽  
Sucrose Density Gradient Centrifugation ◽  
Vicia Faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin–kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin–kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ‘soluble’ RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin–kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.

Alkaline Phosphatase Suhunits in the Culture Filtrate of Pseudomonas aeruginosa

1972 ◽  
Vol 50 (3) ◽  
pp. 268-276 ◽  
Author(s):  
K.-J. Cheng ◽  
D. F. Day ◽  
J. W. Costerton ◽  
J. M. Ingram
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Culture Filtrate ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Growth Cycle ◽  
Active Species ◽  
Acid Dissociation ◽  
Sucrose Density Gradient Centrifugation

Growth of Pseudomonas aeruginosa at pH 6.8 for 14 h under inorganic phosphate limiting conditions is accompanied by the appearance of cell-associated alkaline phosphatase, and no active phosphatase is detected in the culture filtrate. Analysis of cells and the culture filtrate during the growth cycle showed that active phosphatase accumulated simultaneously in both the cells and the filtrate during the early log phase but as growth proceeded beyond 10 h the activity in the filtrate disappeared and the pH dropped from 6.8 to 4.7. Dialysis against 0.01 M Tris and 0.001 M MgCl2 had no effect upon the phosphatase levels of early growth phase (i.e. up to 10 h) culture filtrates. However, dialysis of filtrates obtained after 10 h of growth restored the decreased phosphatase activity to the level present in 10 h filtrates. Treatment of a 14 h filtrate with either trypsin or pepsin prevented the recovery of activity by dialysis whereas such treatments had no effect upon partially purified phosphatase. Sucrose density gradient centrifugation of the 14 h culture filtrate revealed the presence of activity, after dialysis of each fraction against 0.01 M Tris and 0.001 M MgCl2, which corresponded to a molecular weight of 60 000 as compared to a molecular weight of 125 000 for active culture filtrate or partially purified enzyme. Partially purified phosphatase is inactivated and dissociated after treatment at pH 4.2 and the enzyme is reactivated and reassociated after dialysis or dilution into Tris and MgCl2. The results suggest that active phosphatase, molecular weight 125 000, is secreted to the culture filtrate during the early stages of growth and the activity disappears coincidently with the appearance of an inactive species of molecular weight 60 000. The accumulation of the inactive species in the culture filtrate is the result of acid dissociation of the active species as the initial pH of the filtrate decreases from pH 6.8 to pH 4.7.

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