scholarly journals Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

1975 ◽  
Vol 149 (1) ◽  
pp. 65-75 ◽  
Author(s):  
K K Kalghatgi ◽  
P V Subba Rao
Keyword(s):  
Molecular Weight ◽  
Rhizoctonia Solani ◽  
Phenylalanine Ammonia Lyase ◽  
Pathogenic Fungus ◽  
Gradient Centrifugation ◽  
Hill Coefficient ◽  
Velocity Versus ◽  
Kinetic Properties ◽  
Subunit Structure ◽  
Protamine Sulphate

1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (α) and 90000 (β) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Kinetic Properties of Potassium Stimulated ATPase Purified from Gastric Mucosa

1988 ◽  
Vol 43 (7-8) ◽  
pp. 609-612
Author(s):  
Seyhan Tükel ◽  
Turgay İsbir
Keyword(s):  
Gastric Mucosa ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Glycerol Solution ◽  
Enzyme Preparations ◽  
Optimum Activity ◽  
Menten Constant

In this study, partially purified K+-H+ ATPase from frog gastric mucosa were obtained by using differential and density gradient centrifugation. Optimum activity of K+-H+ ATPase (Vmax), Michealis-Menten constant (Km) and Hill coefficient (h) were found as 83.3 μmol Pi·mg prot-1 h-1, 95.2 μᴍ and 0.91, respectively. Enzyme preparations were more stable in glycerol solutions stored at -40 °C. Minimum activity lost was determined for samples stored in 40% (v/v) glycerol solution at -40 °C for two months.

scholarly journals The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806

1974 ◽  
Vol 143 (3) ◽  
pp. 511-520 ◽  
Author(s):  
U. C. Knopf
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Nucleoside Triphosphate ◽  
Subunit Structure ◽  
Bacterial Cells ◽  
Protein Subunit ◽  
Ph Optimum ◽  
Protamine Sulphate

The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH4)2SO4, protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as β′, β, χ1 and α (mol.wts. 160000, 150000, 98000, and 41000±10% respectively). χ1 is the temporary designation for a protein subunit, which might have the same functions as the σ subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41±1°C and a pH optimum of 8.2±0.1. Mg2+ and Mn2+ are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates.

scholarly journals The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain

1977 ◽  
Vol 161 (3) ◽  
pp. 599-607 ◽  
Author(s):  
J Lumsden ◽  
J R Coggins
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subunit Structure ◽  
Multienzyme Complex ◽  
Shikimate Dehydrogenase ◽  
Affinity Adsorbent ◽  
Dimethyl Suberimidate

A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.

Neutralisation of Heparan Sulphate and Low Molecular Weight Heparin by Protamine

1985 ◽  
Vol 53 (01) ◽  
pp. 086-089 ◽  
Author(s):  
A R Hubbard ◽  
C A Jennings
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Low Molecular Weight Heparin ◽  
Heparan Sulphate ◽  
Activated Partial Thromboplastin Time ◽  
Weight Basis ◽  
Low Molecular Weight ◽  
Protamine Sulphate ◽  
At Iii ◽  
Reference Preparation

SummaryThe neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.

Kinetics of Na+/H+ exchange in vascular smooth muscle cells from WKY and SHR: effects of phorbol ester

1995 ◽  
Vol 268 (1) ◽  
pp. C14-C20 ◽  
Author(s):  
G. Hoffmann ◽  
Y. Ko ◽  
A. Sachinidis ◽  
B. O. Gobel ◽  
H. Vetter ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Wistar Kyoto ◽  
Kinetics Of

The kinetic properties of Na+/H+ exchange were investigated in vascular smooth muscle cells (VSMC) in culture from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Antiport activity was measured in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells after nigericin-induced cytosolic acidification. Studies were performed without (control) and with pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA; 200 nM). Na+/H+ exchange markedly differed between the two strains with lower Hill coefficients [1.56 +/- 0.17 (SE) vs. 2.62 +/- 0.36] and higher maximal activity (Vmax) values (55.85 +/- 5.24 vs. 31.11 +/- 2.38 mmol H+.l-1.min-1) in SHR compared with WKY cell lines. PMA markedly altered the antiport kinetics in WKY VSMC with a decrease in the Hill coefficient (1.75 +/- 0.14) without affecting Vmax (31.88 +/- 1.55 mmol H+.l-1.min-1). In VSMC from SHR, PMA had no effect on the kinetic variables investigated. Thus two kinetic abnormalities are present with respect to Na+/H+ antiport activity in VSMC from SHR compared with WKY, i.e., increased Vmax and decreased Hill coefficient. The observation that PMA does not affect the kinetics of the Na+/H+ antiport in VSMC from SHR suggests a marked degree of antiporter prestimulation in this animal model of genetic hypertension.

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.

scholarly journals Comparison of the kinetic properties of the pyruvate dehydrogenase complex from pig kidney cortex and medulla.

1993 ◽  
Vol 40 (3) ◽  
pp. 411-419 ◽  
Author(s):  
T Pawełczyk ◽  
M S Olson
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Product Inhibition ◽  
Hill Coefficient ◽  
Kidney Cortex ◽  
Kinetic Properties ◽  
Kidney Medulla ◽  
Pig Kidney ◽  
Dehydrogenase Complex

The activity of the pyruvate dehydrogenase complex (PDC) purified from pig kidney medulla was affected by K+, Na+, Cl-, HCO3-, HPO4(2-) and changes in ionic strength. Increased ionic strength influenced the activity of PDC from medulla by decreasing the Vmax and S0.5 for pyruvate and increasing the Hill coefficient. The magnitude of these changes was smaller than the corresponding changes for PDC purified from the cortex. In the presence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), HPO4(2-) (10 mM) and at ionic strength of 0.15 M the S0.5 for pyruvate of PDC from medulla was 117 microM and the enzyme complex was saturated by 1.1 mM pyruvate. Under these conditions the S0.5 for pyruvate of PDC derived from cortex was 159 microM and the enzyme was saturated at 4.5 mM pyruvate. Based on the results presented in this report it is suggested that PDC in kidney medulla may be regulated not only by a phosphorylation/dephosphorylation system and end-product inhibition but also via changes in ionic strength.

scholarly journals In situ Impact of the Antagonistic Fungal Strain, Trichoderma gamsii T30 on the Plant Pathogenic Fungus, Rhizoctonia solani in Soil

2019 ◽  
Vol 68 (2) ◽  
pp. 211-216
Author(s):  
MUHAMMAD ANEES ◽  
MUHAMMAD ABID ◽  
SOBIA CHOHAN ◽  
MUHAMMAD JAMIL ◽  
NADEEM AHMED ◽  
...  
Keyword(s):  
Population Density ◽  
Rhizoctonia Solani ◽  
Pathogenic Fungus ◽  
Suppressive Effect ◽  
Fungal Strain ◽  
Soil Microbiome ◽  
Target Dna ◽  
Wide Range ◽  
First Time

Rhizoctonia solani is a soil-borne fungus causing a wide range of plants diseases. Trichoderma gamsii strain T30 has previously been reported as antagonistic against R. solani. Although there are a few studies about the influence of Trichoderma strains on the R. solani densityin a pathosystem in the presence of plant hosts, this report for the first time comprehensively describes in situ effects of a T. gamsii strain on the population density of R. solani in the soil microcosmic conditions. The population dynamics of R. solani were followed in the autoclaved and non-autoclaved soils in artificially prepared microcosms up to day 25 after co-inoculation with T. gamsii in the variable ratios (R1/T1; R1/T0.1; R1/T0.01 of R. solani/T. gamsii). The population density of R. solani was evaluated by qPCR. In the autoclaved soil, target DNA copies of R. solani increased in the control samples from 1 × 105 to 6.5 × 106. At R1/T0.01, the number of target DNA copies were not significantly changed until day 11; however, it decreased by around five times at day 25. At R1/T0.1 and R1/T1, the number of DNA copies was reduced to 2.1 × 106 and 7.6 × 105 at day 11, respectively and the reduction was as much as 17 times at day 25. In the non-autoclaved soil, the number of the fungal cells decreased at day 25 whether inoculated or not with Trichoderma indicating a general suppression by the soil microbiome. In brief, T. gamsii significantly inhibited the growth of R. solani in the soil in situ and there was a general suppressive effect of the natural microbiome.

scholarly journals Soluble neutral and acid maltases in the suckling-rat intestine. The effect of cortisol and development

1974 ◽  
Vol 144 (2) ◽  
pp. 281-292 ◽  
Author(s):  
G Galand ◽  
G G Forstner
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Heat Sensitivity ◽  
Adult Rats ◽  
Ph Optimum ◽  
Maltase Activity ◽  
Acid Maltase ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
The Relationship

The 100000g supernatants from 13-day-old suckling-rat intestinal homogenates contained 43.5% of the total intestinal maltase activity, compared with 7.1% in weaned adult rats aged 40 days. The soluble maltase activity was separated on Sepharose 4B into two quantitatively equal fractions at pH6.0, one containing a maltase with a neutral pH optimum and the other a maltase with an acid pH optimum. The neutral maltase was shown to be a maltase–glucoamylase identical with membrane-bound maltase–glucoamylase in molecular weight, heat-sensitivity, substrate specificity, Km for maltose and Ki for Tris. The soluble enzyme was induced by cortisol, but the ratio of the soluble to bound enzyme fell during induction. Solubility of the neutral maltase was not accounted for by the action of endogenous proteinases under the preparative conditions used. It is postulated that the soluble neutral maltase is a membrane-dissociated form of the bound enzyme and that the relationship between these two forms is modulated by cortisol. The acid maltase generally resembled acid maltase of liver, muscle and kidney. It was shown to be a maltase–glucoamylase with optimal activity at pH3.0, and molecular weight of 136000 by density-gradient centrifugation. At pH3.0 its Km for maltose was 1.5mm. It was inhibited by turanose (Ki=7.5mm) and Tris (Ki=5.5mm) but not by p-chloromercuribenzoate or EDTA. Some 55% of its activity was destroyed by heating at 50°C for 10min. The acid maltase closely resembled β-glucuronidase and acid β-galactosidase in its distribution in the intestine, response to tissue homogenization in various media, and decrease in activity with cortisol treatment and weaning, indicating that it was a typical lysosomal enzyme concentrated in the ileum.

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