Peanut cotyledons

The most difficult, time-consuming, and frustrating aspect of protein purification is stabilization of the protein in an active form. This difficulty derives from the fact that the entire stabilization process must be reconsidered at each step of purification. For example, the first step of phosphoenolpyruvate carboxylase purification from peanut cotyledons... 

In the course of an investigation into the nature of the biosynthetic intermediate of long chain fatty aldehydes, Hitchcock and James proposed that the (S)-2-hydroxy-fatty acid serves as an intermediate in the a-oxidation of fatty acids (40-41). On the other hand, another mechanism for a-oxidation of fatty acids in peanut cotyledons has been proposed by Shine and Stumpf (34), involving hydroperoxy-fatty acids rather than the hydroxy-fatty acids as a transitory intermediate. However, the hydroperoxy intermediate has not been studied so far in marine algae. 

Newcomb and Stumpf (1952) first described an enzyme system in peanut cotyledons that catalyzed the oxidation of C fatty acids to C i aldehydes with liberation of CO2. In the presence of NAD", the aldehyde is oxidized to the corresponding C i fatty acid which can enter the cycle for further a-oxi-dation (Martin and Stumpf, 1959). The peanut system was described as a peroxidase process for which a H202-generating system was required (Mar-kovetzeta/., 1972). 

A very early metabolic event during imbibition, occurring in less than 15 min, appears to be the reformation of keto acids from amino acids by deamination and transamination reactions [37]. Keto acids important for respiratory pathways (e.g. a-ketoglutarate and pyruvate) may be absent from dry seeds such as wheat [70] and peanut cotyledons [138]. They are known to be chemically unstable and it has been suggested [37] that they are stored in the dry seed as the appropriate amino acid, and then reformed on rehydration. This might occur also in barley embryos, seeds of Sinapis alba, and axes and cotyledons of Phaseolus vulgaris [36, 37]. 

Table 5.4. Comparison of various constitutive and functional changes in peanut cotyledon mitochondria occurring after imbibition has taken place...

In peanut cotyledons DNA levels may double up to the 7th-10th day after imbibition even though no cell division seems to occur they decrease thereafter over several days [30, 77]. Part of this increase in DNA could be due to synthesis of mitochondrial DNA (Table 5.4), although it appears that this cannot account for more than 1-2% of the new synthesis [19]. Thus, most DNA synthesis is probably nuclear in origin. The reasons for such synthesis in the absence of cell division are obscure—amplification of genes for enzymes involved in reserve degradation is an attractive, but wholly unsubstantiated suggestion. 

Since fats are stored in oil bodies, it is reasonable to expect that the enzyme responsible for their degradation should be closely associated with these structures. During the first 11 days after imbibition a peanut cotyledon may decrease in dry weight from 345 mg to 143 mg, with a concomitant decrease in fat content of 55%. This represents hydrolysis of 9.4 pmoles of triglyceride per cotyledon per day. Even so, less than 1% of the total lipase activity of a peanut cotyledon has been found associated with the oil body and 99% is associated as an acid lipase (pH 4.6) with a particulate fraction [76]. This fraction has been claimed to be mitochondrial, but that is unlikely. The precise location of the acid lipase is still undetermined but it could be associated with the glyoxysomes. 

Fig. 6.12A and B. Variation in glyoxysomal isocitrate lyase ( ) and malate synthetase (o) in (A) isolated peanut cotyledons incubated in darkness and (B) of these enzymes plus catalase ( ) in the glyoxysomal fraction from the scutellum of germinated maize Zea mays). After Longo and Longo, 1970 [85]... 

Subsequently Giovanelli and Stumpf (107) reported the conversion of propionate-l-C to /9-hydroxypropionate in peanut cotyledon mitochondria. The cofactor requirements were such as to suggest that propionyl CoA is formed. These authors found that the C-1 of the propionate was oxidized to CO2. This suggests that the /9-hydroxypropionate was further oxidized to malonylsemialdehyde and then to CO2 and acetyl CoA. 

Glucose-0 or fructose-C (both uniformly labeled) Acetate-l-C Maturing peanut cotyledon slices Rats Fatty acids Saturated fatty acids of liver 6% of added hexoses converted to fatty acids (223o) (212) ... 

Newcomb, E. H., and Stumpf, P. K., 1952, Fatty acid synthesis and oxidation In peanut cotyledons, "Phosphorus Metabolism, II," W. D. McElroy and B. Glass, eds., p. 291. Johns Hopkins Press, Baltimore. 930pp. 

Gientka-Rychter, A. and Cherry, J. (1968). De novo synthesis of isocitrase in peanut cotyledons. Plant Physiol., 43, 653-659. 

Until 1973 it seemed that there must be at least two different pathways for a-oxidation depending on the source of the enzymes. The pathways that had been studied in pea leaves and in germinating peanut cotyledons apparently had different cofactor requirements and different intermediates seemed to be involved. The discrepancies have now been resolved and a unified pathway has been proposed by Stumpf and his team in California (Figure 3.26). 

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