scholarly journals Salinity endurance of marine macro Rhodophycean algae with special emphasis on myo-inositol biosynthesis: An enzymological analysis from Halymenia venusta Børgesen

2021 ◽  
pp. 30-39
Author(s):  
Dibyendu Sekhar Mahanty ◽  
Sautrik Basu ◽  
Jukta Adhikari
Keyword(s):  
Divalent Cations ◽  
Deae Cellulose ◽  
Monovalent Cations ◽  
Present Communication ◽  
Inositol 1 ◽  
Marine Plants ◽  
Stress Relieving ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Intertidal Habitats

Altered salinity is one the most important perils encountered by marine plants inclusive of algae. Under hyper saline condition plants accumulate several stress relieving osmolytes including myo-inositol, the most widespread cyclitol in plants. The present communication reports the occurrence of myo-inositol biosynthesis in six different Rhodophycean seaweeds growing under stressful intertidal habitats of the Okha coast (Gujarat, India), on the basis of a study conducted on two marker enzymes of myo-inositol biosysnthesis [L-myo-inositol-1-phosphate synthase and D/L-myo-inositol-1-phosphate phosphatise]. Both enzymes were partially purified from Halymenia venusta to about 27 and 39 folds respectively over the homogenate following low-speed centrifugation, 30-75% ammonium sulphate fractionation, successive chromatography through DEAE-cellulose / CM-Cellulose, Sephadex G-200 and BioGel 0.5m / UltrogelAcA 34 columns. The temperature and pH optima for both the enzymes were similar and were recorded to be 350C and 7.5 respectively. For MIPS, D-glucose-6-phosphate and NAD were the exclusive substrate and coenzyme respectively and D/L-MIP was the sole substrate for MIPP. The Km values for D-glucsoe-6-phosphate and β-NAD were recorded to be 3.599 mM and 0.2366 mM respectively, while the Km value for D-MIP was found to be 0.4070 mM. Monovalent cations K+ had slight stimulatory, Li+ was strong inhibitory for both the enzymes. Divalent cations Ca2+ exhibited slight stimulatory and Cd2+ reduced MIPS and MIPP activities. MIPP was stimulated by Mg2+. Cu2+ and Hg2+ were strong inhibitors of both the enzymes. A steady and proportionate increase in the content of free myo-inositol was observed along with elevated levels of recorded salinity.

scholarly journals An inducible hydrolase from Aspergillus niger, acting on carbon–carbon bonds, for phlorrhizin and other C-acylated phenols

1970 ◽  
Vol 116 (5) ◽  
pp. 889-897 ◽  
Author(s):  
T. Minamikawa ◽  
N. P. Jayasankar ◽  
B. A. Bohm ◽  
I. E. P. Taylor ◽  
G. H. N. Towers
Keyword(s):  
Aspergillus Niger ◽  
Metal Ion ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Protamine Sulphate ◽  
Carbon Carbon Bonds ◽  
Alkaline Conditions ◽  
Km Value ◽  
Ammonium Sulphate Fractionation

1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent Km value at pH7.2 was about 0.3–0.4mm for phloretin and 0.15mm for 3′-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3′-methylphloracetophenone, phloracetophenone and 2′,4,4′-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

Cation exchange in cell walls of gram-positive bacteria

1976 ◽  
Vol 22 (7) ◽  
pp. 975-982 ◽  
Author(s):  
Robert E. Marquis ◽  
Kathleen Mayzel ◽  
Edwin L. Carstensen
Keyword(s):  
Cation Exchange ◽  
Cell Walls ◽  
Divalent Cations ◽  
Teichoic Acid ◽  
Dry Weight ◽  
High Ionic Strength ◽  
Monovalent Cations ◽  
Anionic Sites ◽  
Gram Positive Bacteria ◽  
Anionic Groups

The relative affinities of various cations for anionic sites in isolated, bacterial cell walls were assessed by means of a technique involving displacement of one cation by another. The affinity series determined was [Formula: see text]. High affinity was correlated with low mobility of the bound ions in an electric field. The net cation-exchange capacities of walls isolated from a variety of bacteria were estimated by preparing the magnesium forms of the walls, washing them well with deionized water to remove supernumerary ions, and then completely displacing the magnesium with Na+ or H+. Total amounts of magnesium displaced varied from 73 μmol per gram dry weight, for walls of the teichoic acid-deficient 52A5 strain of Staphylococcus aureus to about 520 μmol per gram for Bacillus megaterium KM walls. The amount of displacable magnesium was inversely related to the physical compactness of the walls, except for walls of Streptococcus mutans GS-5. It was found that magnesium or calcium ions can each neutralize, or pair with, two anionic groups in walls suspended in ion-deficient media. Previous work had indicated that these ions may pair with only one anionic group at high ionic strength. Therefore, it appeared that there is a great deal of flexibility in the arrangement of charged groups in the wall. It was concluded also that for cells growing in commonly used laboratory media, which generally contain large excesses of monovalent versus divalent cations, there is a mix of small, cationic counterions in the wall and that monovalent cations may predominate even though the wall has higher affinity for divalent ions.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

The role of sodium chloride in the process of induction by lithium chloride in cells of the Rana pipiens gastrula

Development ◽  
1968 ◽  
Vol 19 (3) ◽  
pp. 387-396
Author(s):  
Lester G. Barth ◽  
Lucena J. Barth
Keyword(s):  
Sodium Chloride ◽  
Culture Medium ◽  
Rana Pipiens ◽  
Divalent Cations ◽  
Pigment Cells ◽  
Distilled Water ◽  
Sodium Ions ◽  
Monovalent Cations ◽  
High Concentration

A study of the effects of a series of monovalent cations, Li+, Na+ and K+, and a series of divalent cations, Mn2+, Ca2+ and Mg2+, upon small aggregates of cells taken from the presumptive epidermis of Rana pipiens gastrulae revealed that these ions induce nerve and pigment cells (Barth, 1965). The effectiveness of both series of ions as inductors was similar to their effects on decreasing the electrophoretic mobility of DNA as determined by Ross & Scruggs (1964). When it was found that sucrose in glass-distilled water also would induce nerve and pigment cells the role of ions as inductors came under closer scrutiny. A study of the nature of the induction by sucrose revealed that a relatively high concentration of sodium ions was necessary in the culture medium used after sucrose treatment (Barth, 1966).

scholarly journals Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa

1976 ◽  
Vol 155 (3) ◽  
pp. 607-613 ◽  
Author(s):  
P R Flanagan ◽  
S H Zbarsky
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Large Loss ◽  
Arrhenius Plots ◽  
The Poor ◽  
Km Value ◽  
Soluble Material

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 × 10(-5) M with thymidine 3′-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.

scholarly journals How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

1994 ◽  
Vol 104 (3) ◽  
pp. 425-447 ◽  
Author(s):  
A R Lindsay ◽  
A Tinker ◽  
A J Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Relative Permeability ◽  
Single Channel ◽  
Divalent Cations ◽  
Monovalent Cations ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Conduction Pathway

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60-0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of lipids with intestinal brush border membrane preparations

1982 ◽  
Vol 60 (9) ◽  
pp. 904-909 ◽  
Author(s):  
P. Proulx ◽  
J. McNeil ◽  
I. Brglez ◽  
D. G. Williamson
Keyword(s):  
Brush Border ◽  
Protein Concentration ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Monovalent Cations ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border ◽  
Ph Range ◽  
Assay Conditions ◽  
Lipid Structures

Conditions for uptake of lipids by rabbit intestinal brush border membrane preparations were investigated. A variety of lipids were found to be incorporated, including choline and ethanolamine phosphatides as well as cholesterol, diglyceride, and fatty acid. The incorporation of those lipids tested was enhanced by Ca2+ and other divalent cations but not by monovalent cations. The optimal Ca2+ concentration was approximately 10 mM. The uptake varied with lipid and membrane protein concentration and proceeded at rates which were too rapid to measure under several assay conditions tried. Incorporations were decreased substantially outside the pH range of 6.5–8.0. The effect of one lipid, phosphatidylcholine, on the structural appearance of the membrane fraction was examined by electron microscopy. No free or surface-bound lipid structures could be detected and the membrane fractions appeared to be unchanged after uptake.

The Separation of two Deoxyribonucleases from Extracts of Intestinal Mucosa of the Rat

1972 ◽  
Vol 50 (6) ◽  
pp. 697-703 ◽  
Author(s):  
C. Y. Lee ◽  
Sheilia Lawrence ◽  
S. H. Zbarsky
Keyword(s):  
Intestinal Mucosa ◽  
Ammonium Acetate ◽  
Divalent Cations ◽  
Deae Cellulose ◽  
Sodium Formate ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dnase Ii ◽  
Ammonium Acetate Buffer ◽  
Optimum Activity

Two deoxyribonucleases have been demonstrated in cell-free extracts of rat intestinal mucosa. The enzymes were separated by ion-exchange chromatography on DEAE-cellulose and further purified by partition on hydroxyapatite. One DNase had optimum activity at pH 6.8–7.0 in ammonium acetate buffer, required Mn2+ or Mg2+ for activity, and was inhibited by EDTA. This enzyme is a DNase I by Laskowski's criteria Advan. Enzymol. 29, 165 (1967). The second enzyme was a DNase II with optimum activity at pH 3.5–4.0 in sodium formate buffer, was not inhibited by EDTA, and showed no requirement for divalent cations. Both enzymes were active only with native DNA and had no action on heated DNA or purified yeast transfer RNA.

scholarly journals Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1970 ◽  
Vol 117 (2) ◽  
pp. 215-220 ◽  
Author(s):  
W. P. Hsu ◽  
G. W. Miller
Keyword(s):  
Enzyme Activity ◽  
Nicotiana Tabacum ◽  
Differential Centrifugation ◽  
Enzyme Protein ◽  
Sodium Amalgam ◽  
Low Concentrations ◽  
Nicotiana Tabacum L ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Final Purification

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent Km value of 3.6×10−5m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30μg/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe2+ concentrations up to 0.5mm, beyond which inhibition occurred. Co2+ and Mn2+ were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe3+ and Cu2+, both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.

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