Substrate pectin

Mode of action and substrate specificity of the purified enzyme were determined by following the decrease in viscosity and the increase in absorbance at 235 nm in reaction mixtures in the presence of 0.187 % substrate (pectin or pectate) at pH 8.0. 

The second group can be further subdivided according to the substrate (pectin or pectic acid) and to the site of attack (endo-/exo-enzyme), as shown in Table 4.28. The endo-enzymes strongly depolymerize and rapidly reduce the viscosity of a pectin solution. 

The substrate for HGA synthesis is UDP-D-galacturonic acid (UDP-GalA) (81). UDP-GalA has been isolated from plants (59) and several pathways for its synthesis in planta have been reported (31). In vitro studies on pectin biosynthesis have been hampered since radiolabeled UDP-galacturonic acid is not commercially available. Previous researchers have used several... 

Most cell walls are able to deesteriJfy exogeneous pectin very rapidly but in vivo these cell walls still contain large amounts of methyl-esterified pectins. How can the substrate and the enzymes coexist inside cell walls ... 

With endogeneous pectic polysaccharides as substrates, the pectin methyhransferase activity was measured as radioactivity linked to oxalate-soluble polys x harides after extensive washing of microsomes with IM ethanolic NaCL Figure 2 shows that the rate of methylesterification of pectic substances was maximal on days 4 and 6 these maximum activities were observed within this period in at least five independent ejqjeriments. On the other hand, little activity was noted in young cells before day 2, and in old cells after day 9. In other words during the stationary phase the newly synthesised pectins remained unesterified because of the lack of pectin methyltransferase activity. 

In these experiments the pectin methyltransferase activity in vitro was generally only a few picokatal g" of protein, due mainly to the limited amount of SAM and endogeneous polysaccharide substrate. 

However the addition of exogeneous pectic substrate increased the incorporation of radioactivity up to twelve-fold. At neutral pH, the stimulation was larger in the presence of highly methylated pectin than in the presence of polygalacturonic acid. At pH 5.5, on the other... 

HPAEC analyses were carried out to determine the oligomeric products released from various pectic substrates after depolymerization by the PL isoenzymes. Action pattern analyses for the concerted action of PL isoenzymes utilized 68% esterified pectin as substrate. One-ml reaction mixtures in a buffer system as detailed in section 2.2. comprising 0.5% (w/v) substrate and 5 U of enzyme were incubated for 30 s to 18 h, and then thermoinactivated. Samples of 750 pi were applied to a Carbopac PA-1 (Dionex) column before the carbohydrates were eluted over a period of 70 min using a gradient of 0.2 M KOH, 0.05 M K-acetate to 0.2 M KOH, 0.7 M K-acetate. Detection employed a Pulsed Electrochemical Detector (PED, Dionex) in the integrated amperometry mode according to the manufacturer s recommendations. 

Intrigued by the finding that Eca PLs exhibit notable differences in their kinetics, HPAEC analyses were carried out to examine the products from the depolymerization of PGA and 31% esterified pectin. After 18 h of incubation with PGA, PL1 and PL2 had produced mainly di- and trimers. Similariy, main products of PL3 action were trimers, followed by dimers. Moreover, it was the only enzyme found to produce monomers from unesterified substrates with a degree of polymerization >3. Using 31% esterified pectin as a substrate, similar end products were released by the PLs as from PGA. In addition to the products described, traces of tetra- up to octamers were detectable. While PL1 and PL2 released di- and trimers at almost... 

More detailed information was obtained from inspection of the time-course for product formation following degradation of pectin with enzyme combinations. Supplementation with PL1 and PL2 together caused high initial activities followed by a significant reduction after around 150 s. Further addition of PL1 or PL2 after 160 s effected no increase in product formation, probably due to exhaustion of available substrate. Alternatively, supplementation with PL3, either initially or after 160 s, stimulated a pronounced enhancement of pectinolysis. 

Since the first steps of pectin degradation are extracellular, the intracellular catabolism is dependent on the permeabihty of the membrane to the external substrates. Oligogalacturonides can enter the cells but the corresponding transport system(s) have not yet been identified (14). Two transport systems which mediates entry of monomers were characterised ExuT for galacturonate uptake (35) and KdgT for DKI, DKIl or KDG uptake (36) (Figure 1). 

---