Arginine Decarboxylase of Oats Is Clipped from a Precursor into Two Polypeptides Found in the Soluble Enzyme

Abstract

We have examined soluble oat (Avena sativa) arginine decarboxylase by probing its structure with polyclonal antibodies that separately recognize amino-terminal and carboxyl-terminal antigens and with a monoclonal antibody that immunoprecipitates enzyme activity. These experiments indicated that oat arginine decarboxylase is clipped from a 66,000-D precursor polypeptide into 42,000-and 24,000-D produce polypeptides. Both of these are found in the enzyme and may be held together by disulfide bonds. A full-length precursor protein could not be detected in plants but could be produced by expression of the cDNA in Escherichia coli. Analysis of the expression of the cDNA in E. coli, with antibodies and using pulse labeling with [35S]methionine, indicated that the bulk of the expressed protein was the full-length 66,000-D form. Small amounts of 42,000-and 24,000-D polypeptides could also be detected. A reconstruction experiment, adding a radioactively labeled full-length protein isolated from E. coli to powdered oat leaves, supported the idea that the protein extraction method used for western blots was not likely to result in artifactual proteolytic degradation.Putrescine synthesis in higher plants can proceed via two alternative pathways from ornithine via ornithine decarboxylase and from arginine via arginine decarboxylase (EC 4.1.1.19) through the intermediate agmatine. The arginine decarboxylase pathway is also present in some bacteria, including Escherichia coli (15), but is largely absent from other eukaryotic kingdoms. Animals and fungi rely predominately on ornithine decarboxylase as the initial, highly regulated, step in polyamine synthesis. In previous research, we (10,11) for the oat leaf arginine decarboxylase. The cloning strategy involved purifying the arginine decarboxylase polypeptide, N-terminal amino acid sequencing, construction of a degenerate oligonucleotide probe, and screening of an oat leaf cDNA library. Extensive similarity exists between the oat and E. coli arginine decarboxylase amino acid sequences. We also generated an antibody (anti-C in Table I) to a carboxyl-end portion of the arginine decarboxylase polypeptide. Analysis of the cDNA and oat leaf proteins indicated that arginine decarboxylase of oats might be proteolytically processed (1) based on the following considerations: The arginine decarboxylase cDNA contains an open reading frame that encodes a 66,000-D polypeptide; however, experiments probing oat leaf extracts with the anti-C antibody revealed 24,000-and 34,000-D polypeptides on western blots. The 24,000-D form was the same size as the polypeptide we (2) characterized via its ability to bind the enzyme-activated irreversible inhibitor DFMA3. The amino terminus of the 24,000-D DFMA-binding polypeptide we purified was subsequently found internally in the predicted arginine decarboxylase open reading frame, approximately two-thirds of the way from the amino to the carboxyl end. The results of both the purification of DFMA-binding activity and the probing of plant extract...

Cloning of Tomato (Lycopersicon esculentum Mill.) Arginine Decarboxylase Gene and Its Expression during Fruit Ripening

“…In animal systems, the regulation of ODC and SAMDC expression at translational/postrranslational level has been well documented, and polyamines themselves have been shown to exert control over rates of translation of ODC and SAMDC mRNAs as well degradation of ODC and SAMDC proteins (Heby and Persson, 1990). In oat, it has been shown that the ADC polypeptide is posttranslationally processed (Malmberg et al, 1992), but whether this processing is related to enzyme activation is not clear. Whether the tomato ADC protein, like the oat protein, is also processed in vivo is not known at this stage, and further work is needed to address this question.…”

Molecular characterization of the Arginine decarboxylase gene family in rice

“…Although many dicot Adc genes are available in the databases, only two monocot representatives have been isolated: those from oat (Bell and Malmberg 1990) and rice (Akiyama and Jin 2007). The oat Adc cDNA encodes a 66-kDa precursor that may be proteolytically processed into 42 and 24 kDa products (Malmberg et al 1992;Malmberg and Cellino 1994). The inhibitor a-difluoromethylarginine (DFMA) binds to the C-terminal 24-kDa fragment, indicating that at least part of the active site resides there (Malmberg and Cellino 1994).…”

Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress

“…The translation product of the adc mRNA is an inactive precursor protein with a molecular mass of Ϸ60 kDa. This precursor is cleaved to produce an N-terminal fragment and a 24-kDa C-terminal fragment containing the ADC active site (40,41). This active ADC form catalyzes the decarboxylation of arginine, leading to the accumulation of putrescine.…”