Plant enzyme isolation

CELL WALLS AND SECONDARY PRODUCTS AS OBSTACLES TO PLANT ENZYME ISOLATION PROBLEMS AND SOLUTIONS, INCLUDING A SIMPLE LIQUID-NITROGEN HOMOGENIZER FOR BULK TISSUE EXTRACTION ... 

Control of pH is especially important in plant enzyme isolation. As a generalization, low pH is desirable. Phenolic compounds, which are ubiquitous in plants, ionize at high pH, and this makes them subject to spontaneous air-oxidation (and subsequent covalent-addition reactions). Ionized phenolics will not form H-bonded complexes either with proteins or with adsorbent polymers. The former might be desirable, but the latter is not. A principal target of covalent addition reactions with proteins is the un-ionized -NH2 group. This applies to 1, 4-addition by quinones md sesquiterpene lactones and to the isothiocyanate addition reaction. Low pH converts amino groups to the unreactive ionized form. Obviously the general factors of protein chemistry also apply, such as possible denaturation or isoelectric precipitation. 

We have reviewed literature and experimental results which demonstrate that the plant cell wall is a greater obstacle to plant enzyme isolation than has been appreciated. We have also discussed the use of cryogenic techniques in combatting both secondary metabolites and cell walls, and described a convenient liquid-N2 homogenizer for bulk extraction of plant tissues. In addition we have pointed out that plant phenolics are not the only secondary products that cause problems in plant enzymology. 

Adsorbent polystyrene as an aid in plant enzyme isolation. Phytochemistry 18 1049 (1979). 

Plant. In rice plants, propanil is rapidly hydrolyzed via an aryl acylamidase enzyme isolated by Still (1968) forming the nonphototoxic compounds (Ashton and Monaco, 1991)... 

A characteristic feature of the SuSy isoforms is a conserved phosphorylated serine residue near the N-terminus [8-10]. In-vivo studies have demonstrated that phosphorylation and dephosphorylation direct the distribution of SuSy isoforms in the plant cell [10-12]. The soluble phosphorylated SuSy interacts with the actin cytoskeleton in the cytoplasm [13], and the dephosphorylated SuSy isoforms are targeted to the cell membrane to form complexes with other enzymes, e.g., glucan synthase, catalyzing cellulose biosynthesis from sucrose [4, 10, 14]. In this respect, recent studies on the dephosphorylated enzymes by cloning and expression of SMS genes in E. coli have shown differences in some biochemical features when compared to the enzymes isolated from the corresponding plant material. Recom-... 

Desaturation takes place in a stepwise fashion, and many intermediate compounds with fewer double bonds are known (Eq. 22-10).118/121-123 The enzymes required have not been characterized well until recently. Plant enzymes are present in small amounts, and isolation has been difficult. However, the genes for carotenoid biosynthesis in such bacteria as the purple photosynthetic RhodobacterRhodospirillum,125 and Rubrivarax,126 the cyanobacterium Synechococcus,127 and the nonphotosynthetic Erwinia44/118 have been cloned, sequenced, and used to produce enzymes in quantities that can be studied. Matching genes from higher plants have also been cloned and expressed in bacteria.123... 

The fructose bisphosphatase of green plants has an amino acid sequence which is very similar to those of the corresponding enzymes isolated from other sources such as yeast or mammals, except that the plant enzyme has an additional sequence of 20 or so amino acids that has no counterpart in the enzymes found in the other species. What function might this additional sequence have in the plant enzyme ... 

Bentley and Bhate (66) described the properties of the enzyme isolated from P. notatum, and Wallenfels and Herrman have reported the isolation and substrate specificity of a mutarotase from E. coli (63). The widespread occurrence of the enzyme in higher plants (53), fish (69), birds (70) and amphibia (69) has been noted. Sacks has recently described the properties of the enzyme in human red blood cells (75). 

These polysaccharides have been synthesized in vitro from various sugar nucleotides with enzymes isolated from plant sources. 

Only one of the three FDPases was found to be sensitive to inhibition by AMP this was the neutral FDPase present in ungerminated castor beans. During germination a second activity appeared which was also active with RuDP. The alkaline activity was present in mature photo-synthesizing leaves, and its properties resembled those of the enzyme isolated from spinach leaves by Racker and Schroeder (55). All of the enzymes from the castor bean plant were found to require either Mg2+ or Mn2+. 

The fact that the activity of isolated respiratory enzymes isolated mitochondria, one-celled organs and organs of plants has... 

The existence of sulfite oxidase in plants snch as Arabidopsis thaliana (wall or thule cress), tobacco, pea, spinach, barley, carrot, and poplar trees has now been confirmed, and the enzyme from A. thaliana has been structurally characterized. The homodimeric, peroxisomal enzyme lacks the heme domain observed in animal sulfite oxidases, making it the simplest Mo-MPT enzyme yet isolated from eukarya. The animal and plant sulfite oxidases have an overall 47% sequence identity. The detoxification of sulfite, produced in the assimilation of sulfate into sulfur-containing amino acids and membrane components (sulfatides) and from environmental sources (acid rain), is also the principal role of the plant enzymes. 

In general, plant biosynthetic pathways are understood poorly when compared with prokaryotic and fungal metabolic pathways. A major reason for this poor understanding is that genes that express complete plant pathways typically are not clustered together on the genome. Therefore, each plant enzyme often is isolated individually and cloned independently. However, several enzymes involved in plant alkaloid biosynthesis... 

Coffee and tea plants seem to contain a variety of N-methyltransferase enzymes that have varying substrate specificity (168, 169). For example, a caffeine synthase enzyme isolated from tea leaves catalyzes both the N-methylation of N-methylxanthine and theobromine (176). The substrate specificity of the methyltransferases can be changed by site-directed mutagenesis (177), and the crystal structure of two of the N-methyltransferases has been reported recently (178). 

Phosphomevalonate kinase (PMK, EC 2.7.4.2) is the enzyme involved in the second step of the terpenoid biosynthesis and catalyzes the reversible conversion of mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP, a key step in that synthesis. Fig. (6), [289-290]. Kinetic characterisation of PMK has been carried out using enzymes from mainly animal sources including human, S. cerevisiae and some plants. In addition, PMK has only been partially purified and characterized. It seems that this enzyme is quite a unique enzyme, but its characteristics reveal some remarkable differences among diverse sources. Thus, pig liver and human liver PMKs show molecular weights between 21 and 22 kDa [291-292], whereas the enzyme isolate from S. cerevisiae has a molecular weight of 47 kDa [293]. 

An in vitro assay for intrinsic sulfonylurea activity has been developed using isolated plant enzyme (5). The Icn for ALS inhibition is defined as the concentration of sulfonylurea that... 

EPSPS has been isolated from both microorganisms and plants, and several of its properties have been studied. The bacterial and plant enzymes are mono functional with molecular mass of 44-48 kD (8-15). The fungal enzyme is a part of the multifunctional arom complex which catalyzes four other reactions of the shikimate pathway (16). While the bacterial enzymes show differences with respect to glyphosate sensitivity, the plant enzymes exhibit a much more narrow range of sensitivity (17). This accounts for the susceptibility of most plant species to glyphosate. 

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