Inhibition of Plant Lactate Dehydrogenase Isoenzymes by Benzoic Acid and Cinnamic Acid Derivatives

Summary Several phenolic compounds such as derivatives of benzoic and cinnamic acid were investigated with respect to their inhibitory effect on potato tuber lactate dehydrogenase isoenzymes. Ki values were determined and it was found that they were in the range of 10−2 M for several benzoic acid derivatives while they were in the range of 10−5 M for several hydroxylated cinnamic acid derivatives such as 3,4-dihydroxy cinnamic acid, p(m)nitrocinnamic acid, and 7,8-dihydroxy-4-methyl coumarin as well as kynurenic acid. Also coumaric acid derivatives revealed a very strong inhibition: The K1 values for coumarin and p-coumaric acid were in the range of 10−4 M. The inhibition by all aromatic co…

Genetic variation of European beech (Fagus sylvatica L.) along an altitudinal transect at mount Vogelsberg in Hesse, Germany

Allelic and genotypic variation at 13 different enzyme loci of autochthonous European beech (Fagus sylvatica L.) was investigated in six 110-160-year-old stands growing at elevations between 150 and 660 m above sea level on the western slope of mount Vogelsberg in central Germany. The highest elevated population showed the highest number of effective alleles (Ne), the highest total heterozygosity (He) and the highest population differentiation deltaT. Also, the genotype SKD-A2A3 of shikimate dehydrogenase was significantly more frequent at the two highest elevated stands (P = 11%) than at the three lowest elevated stands (P = 1%). Further differences in genotype frequencies between 11 of 15…

Determination of molecular weights and Stokes' radii of non-denatured proteins by polyacrylamide gradient gel electrophoresis. 1. An equation relating total polymer concentration, the molecular weight of proteins in the range of 104-106, and duration of electrophoresis

Untreated and processed gel plates of polyacrylamide (PAA) gradient gels were cut into strips perpendicularly to their length, and the wet and dry matter of the sections was determined. In untreated gels the apparent dry matter, as well as the relative dry matter, are a linear function of the gel length. In processed gels, however, only the apparent gel concentration increases linearly with the gel length, whereas the relative dry matter increases linearly with the square root of the gel length. The %T (content in polyacrylamide) was calculated from the apparent dry matter. The gel gradients used were found to be linear with respect to %T. Six different calibration proteins were run and the…

The Effect of Light on the Growth of Pea Plants and the Subsequent Influence in Shikimate Oxidoreductase (EC 1.1.1.25) Activity

Summary Pea plants were cultured in white light, red and far red light, and in the dark during a period of three weeks. At several states of development we investigated the activity of the enzyme shikimate oxidoreductase, the amount of fresh and dry matter, and the contents of protein in stem, leaves, cotyledons, and roots. The enzyme activity was found to be distributed organ-specifically and uninfluenced by the phytochrome system, but it was significantly depressed in plants grown in the dark compared to plants grown in white light. Enzyme activity occurred also in non photosynthetic plants. Regarding the different light conditions the activity of shikimate oxidoreductase was found to cor…

Intracellular compartmentation and regulation of two shikimate dehydrogenase isoenzymes in Pisum sativum

Summary Pea seeds as well as sprouts and roots contain two isoenzymes of shikimate dehydrogenase. Both isoenzymes can be separated by Polyacrylamide gel electrophoresis as well as through ammonium sulfate fractionation. The molecular weight of both isoenzymes are the same although the net electric charge is different. The Km value for isoenzyme 1 is 3,5 × 10 −4 and the Km value for isoenzyme 2 is 1,67 × 10 −4 M. 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, gallic acid, anthranilic acid and p-methoxycinnamic acid inhibited both isoenzymes competitively. Anthranlic acid showed the largest affinity to both isoenzymes. M-methoxycinnamic acid and m-nitrocinnamic acid inhibited both isoe…

Extraction of Enzymes from Tissues, Cells and Cell-Organelles

Microorganisms, such as bacteria, algae, moulds and others, are ruptured by sonication, by passage through a French press [1, 2] (Fig. 2.1) or a Manton-Gaulin homogenizer [3], by blending with glass beads [4], or by digesting the cell walls enzymically [5]. Extract preparation is preferably performed in the cold (+4 °C).

ELECTROPHORESIS | Porosity Gradient Gels

Determination of molecular weights and Stokes' radii of non-denatured proteins by polyacrylamide gradient gel electrophoresis 3. Estimation of the upper and lower size limits of carbonic anhydrase as a model for complexing enzymes

Studying the separation behavior of various native carbonic anhydrase isozymes from mammalian erythrocytes we found that the migration of these enzymes differs from that of the marker proteins commonly used in gradient gel electrophoresis. In alkaline buffer systems the enzymes from human, bovine, rabbit, and canine erythrocytes start to migrate with a size apparently 6 to 12 times larger than their monomeric size, then gradually lose in apparent size and finally end up in a size equivalent to their monomeric mol mass. We determined the monomeric mol mass of the various carbonic anhydrase forms to be 23 000 to 39 000 (g/mol). These values are in accordance with different data in the literat…

Determination of molecular mass, Stokes' radius, frictional coefficient and isomer-type of non-denatured proteins by time-dependent pore gradient gel electrophoresis.

Molecular mass, Stoke's radius, frictional coefficient and isomer-type of non-denatured proteins can be obtained by time-dependent gradient gel electrophoresis by evaluating the resulting data using a two-step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non-denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from current-year needles of spruce. The resulting data as well…

Molecular size and net charge of pathogenesis-related enzymes from barley (Hordeum vulgare L., v. Karat) infected withDrechslera teres f. teres (Sacch.) Shoem

Molecular size and net charge of isoforms of pathogenesis-related (PR) chitinase, beta-1,3-glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., v. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time-dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL-MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703-709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxid…

Sodium Dodecylsulphate Electrophoresis

Sodium dodecylsulphate (SDS) consists of an aliphatic chain of 12 C atoms to which at one end a sulphate residue is bound. It forms complexes with both the polar and non- polar amino acid residues of proteins irrespective of their sizes and shapes, leaving the primary structure uninfluenced. In electrophoresis SDS is used: a) to separate (enzyme) proteins into their monomeric constituents, b) to estimate the molecular mass of unfolded (and reduced) polypeptides, and c) to keep membrane proteins in a solubilized state.

Applicability of the log MM - √D relationship to linear polyacrylamide gradient gel electrophoresis under a wide range of experimental conditions

Recently we reported about a linear correlation between the logarithm of the size of native proteins (log mol mass or log Stokes' radius) and the square root of their migration distance (- √D) in linear polyacrylamide (PAA)-gradient gels (G. M. Rothe and H. Purkhanbaba, Electrophoresis 1982, 3, 33–42). The linearity between log MM and √D is not subject to time using homogeneous buffers in electrophoresis, no matter how the constants of the corresponding regression lines, slope and intercept change as a function of time. The realiability of this correlction has been re-examined with 0.7 mm thin gel plates and extending the time of electrophoresis under non-denaturating conditions from 2 to 9…

Nutrient element and carbohydrate status of Norway spruce at Mt. Kleiner Feldberg in Taunus exposed to air pollution and soil acidification

Affinity chromatographic separation of plant lactate dehydrogenase

Abstract Lactate dehydrogenase from potato tubers was purified by the use of several standard purification procedures as well as by affinity chromatography on C

Methods for Separating Native Enzymes

In the course of electrophoresis the stability of an enzyme depends on such conditions as (a) pH-value, (b) ion strength and ion species, (c) effector molecules, (d) temperature and (e) properties of the separation matrix. These parameters were empirically optimized for starch gel electrophoresis [1–3] and cellulose acetate electrophoresis [4, 5] when analyzing predominantly animal and human specimen. A major advantage of these types of separation media is that practically every buffer system can be used to separate enzymes whereas in disc-gel electrophoresis [6–8] the number of applicable buffer systems is limited. When using isoelectric focusing to separate native enzymes no buffer choice…

Preparation of extracts from mature spruce needles for enzymatic analyses

It was possible to extract simultaneously several active enzymes involved in the carbohydrate or the amino acid metabolism from spruce needles [Picea abies (L.) Karst.] when a) a 100 mM Na-Pi buffer of pH 7.5 containing 5% PVPP and 0.5% Triton X-100 was used and when b) the resulting crude extracts were freed from lowmolecular-weight compounds by gel-chromatography using the separation medium Fractogel TSK HW-40. Besides Triton X-100, Triton X-305, Myrij-52 and Brij-35 were tested, but 0.5% Triton X-100 brought about the most active enzyme extracts. In crude extracts prepared from spruce needles during the early summer a high increase in absorbance at 334 nm was observed when the co-substra…

The Influence of Thiol Reagents on the Isoenzyme Pattern of Plant L+Lactate Dehydrogenase (EC 1.1.1.27)

Summary Lactate dehydrogenase from potato tubers was incubated together with various thiol reagents and the effect on the electrophoretic behavior of its isoenzymes studied. while disulfides, alkylating agents, and H202 did not alter the number and electrophoretic behavior of the isoenzymes of L + LDH, reduced glutathione and 2-mercaptoethanol led to an increase in the number of isoenzymes. The reaction was dependent on concentration and pH. On the other hand, cysteine and cysteamine had no such effect. Results are discussed in view of the possible secondary nature of the plant LDH isoenzymes.

Determination of molecular weights and Stokes' radii of non-denatured proteins by polyacrylamide gradient gel electrophoresis. 2. Determination of the size of stable and labile molecular weight variants of enzymes from plant sources

Under certain conditions in polyacrylamide gradient gel electrophoresis (PAGGE), a linear correlation between the logarithm of the size of calibration proteins (log MW or log Rs) and the square root of their migration distance (√D) can be observed; slope and intercept of the calibration curve depend on the duration of electrophoresis; linearity, however, is maintained over a wide range (4-60 h, 200 V) (Rothe and Purkhanbaba, Electrophoresis 1982, 3, 33–42.) Using this method the reaction of plant isozyme systems penetrating a linear polyacrylamide (PAA) gradient gel was investigated: lactate dehydrogenase (LDH) from potato tubers behaves similarly to animal calibration proteins. The enzyme …

Chemistry of Enzyme Visualization

The basic principle of enzyme visualization in situ is to present an enzyme with a solution containing an enzyme specific substrate. Demonstration of an enzyme is achieved if the catalytic action of the enzyme on this substrate produces a coloured reaction product. Often, however, the primary reaction products are colourless and require coupling with a visualizing agent to generate a coloured, preferably insoluble, final reaction product.

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Hoglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as “Piceirhiza nigra” and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).

Data Evaluation in Population Genetics and Evolution

Isozymes maybe generated by different enzyme loci (a) (isoenzymes), (b) alleles of a locus (allozymes) or (c) post-translational modifications (secondary isozymes). Differences in isozyme numbers and isoenzyme properties can be used for evolutionary studies. But quantitations of genetic variation among or within populations are obtainable only from allozyme frequencies.

Computer-aided calculation of the molecular size of nondenatured proteins in pore-gradient gel electrophoresis

A computer program written in Turbo C is described, which uses the two-step mathematical procedure published recently (Rothe, G. M., Electrophoresis 1988, 9, 307-316) to evaluate the molecular mass, Stokes' radius, spherical radius, and frictional coefficient of nondenatured proteins. The program runs on any IBM-PC or 100% compatible IBM-PC, provided the disk operating system MS-DOS or PC-DOS 3.0 or later has been installed. Functions that are permanently in use are accessible by menu. Storage and loading of data from disk and help instructions can be called by use of function keys. The program provides several tables into which inserted and calculated data is automatically integrated. Each…

A Compilation of Protocols to Visualize Enzymes Following Electrophoretic Separation

The following tables supply protocols for the electrophoretic separation as well as for the succeeding visualization of enzymes. Separation systems and enzyme visualization procedures are adjusted to each other.

A study on 3‐deoxy‐D‐arabino‐heptulosonic Acid 7‐Phospate Synthase in Higher Plants. The existence of three isoenzymes in Pisum sativum