Pea seed

As noted above, the presence of Met(O) in proteins would go undetected after acid hydrolysis and subsequent amino acid analysis. Thus, since this method of hydrolysis is most commonly used, it is impossible to ascertain from the literature the abundance of Met(O) residues normally present in proteins. However, a number of studies have reported the presence of Met(O) residues in various proteins using one of the appropriate procedures described above. It has been found that Met(O) residues comprise 30% of the total Met in proteins isolated from bovine glomerular basement membranes and anterior lens . Other investigators have reported that the levels of Met(O) in proteins of the trabecular meshwork of human eyes increased with the age of the donor . The amount of Met(O) detected ranged from 15% (10 years old) to 55% (79 years old) of the total methionine content found in the tissue samples. Other studies have shown that in certain species of clams the proteins of the hinge ligament contain only Met(0) residues and no Met . In addition, it has also been reported that as much as 18% of the Met residues in pea seed proteins is in the form of Met(O) . Lastly, Met(O) residues have been found in... 

In normal human subjects, some 25 % of total body iron (800-1000 mg) is present in the storage forms, mostly as ferritin. Whereas it is likely that all mammalian cell types contain some ferritin, haemosiderin in normal subjects is essentially restricted to cells of the reticuloendothelial system. Ferritin turns out to be almost universal in its distribution ferritin and ferritin-like proteins have been found in all organisms except for one or two archaebacteria. In contrast, haemosiderin has not been found to any extent outside of iron-loaded animals, except for a brief report of a phytosiderin in pea seeds (Laulhere et ah, 1989). 

When pea seed apoferritin is reconstituted in vitro in the absence of phosphate, the reconstituted mineral core consists of crystalline ferrihydrite (Rohrer et ah,1990 Wade et ah, 1993 Waldo et ah, 1995). Conversely, horse spleen ferritin reconstituted in the presence of phosphate produces an amorphous core (Rohrer et ah,1990 St. Pierre et ah, 1996)... 

In this chapter we describe the use of pea seeds to express the bacterial enzyme a-amylase. Bacterial exoenzymes like the heat stable a-amylase from Bacillus licheni-formis are important for starch hydrolysis in the food industry. The enzymatic properties of a-amylase are well understood [13,14], it is one of the most thermostable enzymes in nature and it is the most commonly used enzyme in biotechnological processes. Although fermentation in bacteria allows highly efficient enzyme production, plant-based synthesis allows in situ enzymatic activity to degrade endogenous reserve starch, as shown in experiments with non-crop plants performed under greenhouse conditions [12,15]. Finally, the quantitative and sensitive detection of a-amylase activ-... 

Procedures for Foreign Protein Expression in Transgenic Pea Seeds... 

Tab. 12.1 Numberofindependenttransgenic lines, numberofsingle insertion lines and the rangeof enzyme activities in pea seeds of three commercial varieties obtained under greenhouse conditions.

Fig. 12.2 Enzyme activity during pea seed development under field conditions.

FANG, T-Y., BAISTED, D.J., 2,3-Oxidosqualene cyclase and cycloartenol-S-adenosylmethionine methyltransferase activitites in vivo in the cotyledon and axis tissues of germinating pea seeds, Biochem. J., 1975,150, 323-328. 

Craig S, Goodchild DJ. Periodic acid treatment of sections permits on-grid localization of pea seed vicilin in ER and Golgi. Protoplasma 1984 122 91-97. 

Compositional differences in the pea seeds influence the quality of the end products. Pea flours have been used for protein enrichment of a number of cereal-based products however, undesirable sensory characteristics may limit their use, in spite of improved functional effects in food systems. The production of volatile compounds during cooking and baking of foods with pea supplementation affects their acceptability. Enzyme systems active in unheated pea flours may contribute to their functional properties, but adversely affect the sensory quality of the food. 

Pea starch granules are oval, sometimes fissured, with a diameter of 20-40 ym (13). Molecular and structural characteristics of the two main components of field pea starch—amylose and amylopectin—are important in determining functional properties (25,26). Smooth field pea starch concentrate contains 97.2% starch of which 30.3-37.8% is amylose (9,23,25-27), and wrinkled pea starch concentrate contains 94.8% starch, which is 64% amylose (26). The gelatinization temperature of smooth pea starch is between 64 to 69 C, and that of wrinkled pea starch is greater than 99 C to 115 C. Gelatinization temperature depends on maturity of field pea seed and amylose content (26,27). 

The fact that glycogen phosphorylase can be used to polymerize amylose was first demonstrated by Schaffner and Specht [110] in 1938 using yeast phosphorylase. Shortly after, the same behavior was also observed for other phosphorylases from yeast by Kiessling [111, 112], muscles by Cori et al. [113], pea seeds [114] and potatoes by Hanes [115], and preparations from liver by Ostern and Holmes [116], Cori et al. [117] and Ostern et al. [118]. These results opened up the field of enzymatic polymerizations of amylose using glucose-1-phosphate as monomer, and can be considered the first experiments ever to synthesize biological macromolecules in vitro. 

Results of such experiments with enzymes from the sheep brain, pea seed, and E. coli (87) show that, without exception, the relative rates of exchange are (glutamate < glutamine) > (NH3 = glutamine) > (P( ATP) a (ADP ATP). If the substrate interconversion were the rate-limiting step... 

AMP aminohydrolase, an enzyme relatively specific for AMP, has been observed in reptiles (44), erythrocytes (38), snail (45), unfertilized fish eggs (46), invertebrates (47), a variety of mammalian tissues (20), and a particulate fraction of pea seeds (48). Evidence suggests that the frog muscle AMP aminohydrolase is located within or just beneath the sarcolemma (49). The rabbit skeletal and heart muscle enzymes were found in the cytoplasm and mitochondria (20, Jfi, 50, 51), while the enzyme of kidneys and gills of freshwater fish was located in the cytoplasmic fraction (52). The enzyme occurs in most areas of the rat (53) and rabbit brain (54). The nonspecific enzyme from several microbial sources deaminates adenosine triphosphate (ATP) and adenosine diphosphate (ADP) as well as AMP (see Section V). 

In general, AMP aminohydrolase specificities have not been thoroughly defined perhaps because of difficulties until recently in obtaining pure enzyme. In addition to AMP and dAMP, the muscle enzyme catalyzes the deamination of V -methyl AMP, iV -ethyl AMP, for-mycin-5 -monophosphate, adenosine-5 -monosulfate, adenosine-5 -phos-phoramidate, adenosine, ADP (133), adenosine-5 -phosphorothioate, and 6-chloropurine 5 -ribonucleotide (124) ATP, GMP, CMP, 2 -AMP, 3 -AMP, 3, 5 -cyclic AMP, 3-iso-AMP, V-methyl AMP, toyocamycin-5 -monophosphate, tubercidin-5 -monophosphate, and 6-mercaptopurine-5 -ribonucleotide are not deaminated (133). The elasmobranch fish muscle, carp muscle, and avian brain enzymes appear to be specific for AMP and dAMP (123, 125, 126). Extracts from pea seed and erythrocytes and the purified calf brain enzyme are specific for AMP (48, 131, 134). 

Iodoacetate had no effect on the rabbit muscle enzyme (130, 135) but did inhibit the carp muscle and pea seed enzyme (48, 136). Organic mercurials are also reported to inhibit the enzyme from several sources (48, 125, 126, 130, 136). Except for the preliminary report by Wolfenden et al. (92) that mercurials desensitized the rabbit muscle enzyme to allosteric inhibition by GTP, the role of sulfhydryl residues in AMP aminohydrolase is not understood. 

Figure 2. Response of Little Marvel pea seedings to gibberellic acid and a mixture of gibberellic acid and Ceratonia extract...

Quantitation. Combined GC-SICM has been used mainly for quantitation. For a particular GA the absolute intensity of a characteristic ion in its mass spectrum is related to the amount of GA present, using standards to calibrate the instrument. Frydman et al. (35) used this "external standard method" to measure the levels of a number of GAs throughout the development of pea seeds. An alternative and preferable approach employs... 

Sponsel and MacMillan (36,25) have illustrated the use of GAs labeled with deuterium and tritium to quantitate GAs and also to follow their metabolism. In one experiment (25) they injected a measured amount of [2H][3h]gA29 (both species labeled at the same position) into immature pea seeds. Some seeds were extracted immediately and others at regular time intervals thereafter so that the metabolism of GA29 could be studied. 

---