Biosynthesis of α-Amylase in Vigna mungo Cotyledon

Abstract

In vitro translation of RNA extracted from Vigna mungo cotyledons showed that a-amylase is synthesized as a polypeptide with a molecular mass of 45,000, while cotyledons contain a form of a-amylase with a molecular mass of 43,000. To find out whether the 45,000 molecular mass polypeptide is a precursor to the 43,000 found in vivo, the cell free translation systems were supplemented with canine microsomal membrane; when mRNA was translated in the wheat germ system supplemented with canine microsomes, the 45,000 molecular mass form was not processed to a smaller form but the precursor form was partly processed in the membrane-supplemented reticulocyte lysate system. When V. mungo RNA was translated in Xenopus oocyte system, only the smaller form (molecular mass 43,000) was detected. Involvement of cotranslational glycosylation in the maturating process of the a-amylase was ruled out because there was no effect of tunicamycin, and the polypeptide was resistant to endo-6-H or endo-j,-D digestion. We interpret these results to mean that the 45,000 molecular mass form is a precursor with a signal peptide or transit sequence, and that the 43,000 molecular mass is the mature form of the protein.Numerous investigations have been carried out on the mechanism of a-amylase synthesis and transport in both microorganisms and eukaryotes. In all cases, the enzyme appears to be a secretory protein. Investigations with plants (wheat, barley, and rice) showed that a-amylase is synthesized as a precursor polypeptide. In wheat, the larger precursor was processed to the mature form in Xenopus oocytes and by canine microsomes (2), but not by the RER obtained from aleurone layers (15). In barley, newly synthesized a-amylase is associated with the ER (6, 9). In rice, extensive glycosylation is preceded by proteolytic processing during the synthesis of a-amylase (12). Recently, cDNA complementary to mRNA sequence of wheat and barley a-amylase was obtained, and nucleotide sequencing showed that the enzymes have typical signal sequences at their N-terminal ends (16,17 brane. We found that in the cotyledons, a-amylase is synthesized as a large precursor by membrane-bound polysomes, cleaved to mature form in cell-free translation systems supplemented with canine pancreas microsome, and in Xenopus oocyte system. In addition, mature a-amylase is not glycosylated. Possible mechanisms of intracellular transport of a-amylase in V. mungo cotyledons are discussed. MATERIALS AND METHODSPlant Material for RNA Preparation. We used only detached cotyledons because of their high ability of a-amylase synthesis as described previously (7). Vigna mungo dry cotyledons detached from their embryonic axis were immersed in water for 6 h and then surface sterilized in 1% NaOCI solution for 5 min, rinsed with sterile water, and incubated under aseptic conditions for 4 or 5 d as described previously (7). The cotyledons were harvested, frozen with liquid N2, and stored at -20°C until used.Preparation of Poly A' RNA. All procedures were carried out in asepti...

Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds

“…Membrane-bound and free polysomes were prepared from 3-d-old cotyledons (3 g) essentially following the method of Tomura et al (23). Each of the final preparations was suspended in 40 mm Tris-Cl buffer (pH 8.5) containing 20 mM KCl and 1 mm Mg-acetate.…”

“…In vitro translation in the wheat germ system was carried out as described earlier (22) Immunoprecipitates of translation products were isolated by using a small column of protein A-Sepharose (Pharmacia) (23). After SDS-PAGE of the immunopreciptiates, gels were processed for fluorography (Fuji RX film).…”

Concurrent Synthesis and Degradation of Alcohol Dehydrogenase in Elicitor-Treated and Wounded Potato Tubers

“…Having characterized the levels of ADH in arachidonic acid-treated discs, we then chose the 24 and 48 h time points to test the effect of salicylic acid and anaerobic treatments on ADH levels in potato. We had previously shown these also to induce a rapid accumulation of Adh mRNA in potato discs.…”

“…Arachidonic acid-treated or water-treated control discs were labeled for the final Immunoprecipitations were carried out by a procedure modified from Tomura and Koshiba (24). After incubating 1 mL of plant extract overnight at 4°C with 3 uL ADH antiserum, 20 ,uL of protein A Sepharose CL-4B (Pharmacia) was added (1:1 v/v solution in H20), and the extracts were incubated for 2 h with end-over-end agitation.…”

“…The Adh mRNA was shown to accumulate rapidly in potato discs treated with arachidonic acid (20,22), a fatty acid capable of eliciting phytoalexin synthesis in potato tubers (5 24 h. In water-treated control discs, the transcript was also induced but to low levels. We further identified a number of other elicitor and stress treatments capable of inducing Adh in potato discs (22).…”