scholarly journals Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme

1998 ◽  
Vol 331 (3) ◽  
pp. 929-935 ◽  
Author(s):  
Anette HENKER ◽  
Ilka SCHINDLER ◽  
Andreas RENZ ◽  
Erwin BECK
Keyword(s):  
Isoelectric Focusing ◽  
Spinacia Oleracea ◽  
Specific Activity ◽  
Dissociation Constants ◽  
Protein Band ◽  
Enzyme Substrate ◽  
Spinacia Oleracea L ◽  
Starch Debranching Enzyme ◽  
Ph Range ◽  
The Individual

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1–7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands (‘substrate-induced forms’, numbered I–III) with different specific activities, dissociation constants of the enzyme–substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.

Effects of Ampholyte Dissociation Constants on Protein Separation in On-Chip Isoelectric Focusing

2008 ◽  
Vol 8 (7) ◽  
pp. 3719-3728 ◽  
Author(s):  
Jaesool Shim ◽  
Prashanta Dutta ◽  
Cornelius F. Ivory
Keyword(s):  
Electric Field ◽  
Isoelectric Focusing ◽  
Protein Separation ◽  
Dissociation Constants ◽  
Ph Profile ◽  
On Chip ◽  
Ph Range ◽  
Separation Of Proteins ◽  
Ampholyte Dissociation ◽  
Ph Profiles

Numerical simulations are presented for ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of ΔpKs between 1 and 3.5 are investigated for ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of ΔpK (ΔpK > 3 are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of ΔpK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with ΔpK, while the focusing time goes up exponentially with increasing ΔpK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of ΔpK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.

Rat prostatic acid phosphatase: androgenic control of isoelectric focusing patterns

1983 ◽  
Vol 61 (7) ◽  
pp. 744-749 ◽  
Author(s):  
J. Downey ◽  
D. Mahan ◽  
T. G. Flynn ◽  
C. E. Bird ◽  
A. F. Clark
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Isoelectric Focusing ◽  
Specific Activity ◽  
Electrophoretic Pattern ◽  
Prostatic Acid Phosphatase ◽  
Staining Intensity ◽  
Adult Rats ◽  
Enzyme Specific Activity ◽  
Ph Range

To further characterize the androgen dependence of prostatic acid phosphatase (AP), the isoelectric focusing patterns of enzyme activity have been examined for normal and castrated adult rats and for rats receiving androgen injections. Isoelectric focusing was performed in polyacrylamide gels over the pH range 4–8. Naphthyl phosphate was used as substrate for staining. For normal rats there was a single lysosomal band (isoelectric point (pI) = 7.35 ± 0.04), four closely migrating secretory bands (pI = 5.96–5.63), and an androgen-dependent band (pI = 6.37 ± 0.05) which as yet has not been identified as either lysosomal or secretory. Following castration the secretory bands decreased significantly in staining intensity, the androgen-dependent band disappeared, and two new lysosomal bands (pI's = 7.13 ± 0.03 and 7.00 ± 0.03) appeared. With androgen replacement the latter two bands disappeared, the androgen-dependent band reappeared, and the secretory bands increased in staining intensity but with the most anodic of the four appearing before the others. This suggests that it could be a precursor to the others. The isoelectric focusing patterns of AP activity appear to be a better method of assessing the androgen status of the prostate than are the previously used parameters, namely, enzyme specific activity, degree of inhibition by tartrate, and polyacrylamide gel electrophoretic pattern.

scholarly journals Photosynthesis-Inhibiting Activity of N-(Disubstituted-phenyl)-3-hydroxynaphthalene-2-carboxamides

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4336
Author(s):  
Jiri Kos ◽  
Tomas Gonec ◽  
Michal Oravec ◽  
Izabela Jendrzejewska ◽  
Josef Jampilek
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Spinacia Oleracea ◽  
Photosynthetic Electron Transport ◽  
Inhibiting Activity ◽  
Spinacia Oleracea L ◽  
The Individual

A set of twenty-four 3-hydroxynaphthalene-2-carboxanilides, disubstituted on the anilide ring by combinations of methoxy/methyl/fluoro/chloro/bromo and ditrifluoromethyl groups at different positions, was prepared. The compounds were tested for their ability to inhibit photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-Difluorophenyl)-, N-(3,5-dimethylphenyl)-, N-(2,5-difluorophenyl)- and N-(2,5-dimethylphenyl)-3-hydroxynaphthalene-2-carboxamides showed the highest PET-inhibiting activity (IC50 ~ 10 µM) within the series. These compounds were able to inhibit PET in photosystem II. It has been found that PET-inhibiting activity strongly depends on the position of the individual substituents on the anilide ring and on the lipophilicity of the compounds. The electron-withdrawing properties of the substituents contribute towards the PET activity of these compounds.

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Leona Wagner ◽  
Michael Wermann ◽  
Fred Rosche ◽  
Jens-Ulrich Rahfeld ◽  
Torsten Hoffmann ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Specific Activity ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Kidney Cortex ◽  
Porcine Kidney ◽  
Ph Gradients ◽  
Activity Staining ◽  
Ph Range ◽  
Gel Ph

AbstractIn the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4–6, activity staining resulted in at least 17 isoforms between pH 4.8–6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5–6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

HETEROGENEITY OF LONG ACTING THYROID STIMULATING (LATS) – ACTIVITY, THYROGLOBULIN ANTIBODIES AND THYROID MICROSOMAL ANTIBODIES

1973 ◽  
Vol 73 (3) ◽  
pp. 483-488 ◽  
Author(s):  
F. Adlkofer ◽  
H. Schleusener ◽  
L. Uher ◽  
A. Ananos ◽  
C. Brammeier
Keyword(s):  
Isoelectric Focusing ◽  
Graves Disease ◽  
Thyroid Antibodies ◽  
Thyroglobulin Antibodies ◽  
Ph Range ◽  
Long Acting ◽  
Thyroid Microsomal Antibodies

ABSTRACT Crude IgG of sera from 3 patients with Graves' disease, which contained LATS-activity and/or thyroid antibodies, was fractionated by isoelectric focusing in a pH-range between 6.0 to 10.0. LATS-activity was found in IgG-subfractions from pH 7.5 to 9.5, thyroglobulin antibodies and thyroid microsomal antibodies from pH 6.0 to 10.0. It was not possible to separate LATS-activity from the thyroid antibodies by this technique. The results indicate that LATS and the thyroid antibodies are heterogeneous and of polyclonal origin.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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