Per Villand

Insensitivity of Barley Endosperm ADP-Glucose Pyrophosphorylase to 3-Phosphoglycerate and Orthophosphate Regulation

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 [mu]mol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4[deg]C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.

Is There an Alternative Pathway for Starch Biosynthesis in Cereal Seeds

A hypothesis is presented concerning a putative extra-am yloplastic location of barley seed endosperm ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis. The hypothesis is based both on indirect and direct evidence obtained in our laboratory as well as on data of other investigators. It is proposed that ADP-glucose form ed by the extraamyloplastic enzyme is transported to the am yloplasts via an ADP-glucose carrier in the plastid membrane, and then is utilized by the starch biosynthesizing machinery of these organelles. In addition to the extra-am yloplastic form of AGPase, barley endosperm contains also a second isozyme of AGPase, located in the amyloplasts. Presence of isozymes of AGPase in cereal seed endosperm is consistent with biochemical, molecular and genetic data on starch biosynthesis in this tissue

MOLECULAR CLONING AND EXPRESSION OF THE LARGE SUBUNIT OF ADP-GLUCOSE PYROPHOSPHORYLASE FROM BARLEY (HORDEUM VULGARE) LEAVES

A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.

Plant ADP-glucose pyrophosphorylase--recent advances and biotechnological perspectives (a review).

Abstract Recent advances in studies on plant ADP -glucose pyrophosphorylase (AGP), the key enzyme of starch biosynthesis, are presented. AGP constitutesthe First committed and highly regulated step of starch synthesis in all plan ttissues. The importance of AGP in carbohydrate metabolism and several of its features, such as potent regulation by cellular effectors (3-phosphoglycerate and Pi), an unusual two subunit-types structure, tissue-specific and developmentally-regulated expression, and presence of the AGP -deficient mutants, make it an attractive, but complex, target forbiotechnological manipulations. Some strategies for future research on AGP are discussed.

Molecular cloning and spatial expression of an ApL1 cDNA for the large subunit of ADP-glucose pyrophosphorylase from Arabidopsis thaliana

Abstract A cDNA, A p L 1a , corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AG Pase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3′-ends (3′-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homo-logues for AGPase, and shows ca. 58–78 % identity to homologous proteins from other plants/tissues. The corresponding gene was found (rosette and stem leaves, stems, flowers and fruits), consistent with its critical role in starch synthesis in

Differential Processing of Homologues of the Small Subunit of ADP-Glucose Pyrophosphorylase from Barley (Hordeum vulgare) Tissues

Abstract ADP-glucose pyrophosphorylase (AGPase), a two-gene-encoded enzyme, is the key component of starch synthesis in all plants. In the present study, we have used an E. coli expression system for the (over)production of proteins derived from both full length and specifically truncated cDNAs encoding small subunits of AGPase from seed endosperm (AG Pase-Bl) and leaves (AGPase-B2) of barley (Hordeum vulgare). Based on immunoblot analyses, the molecular mass of the expressed A G Pase-Bl (52 kD) was similar to that from endosperm extracts, whereas the expressed AGPase-B2 (56 kD) was larger than that in barley leaves (51 kD). Expression of truncated cDNAs for both the seed and leaf proteins has allowed for a direct verification of molecular masses that were earlier proposed for mature AGPases in barley tissues. The data suggest that seed AGPase-B1 does not undergo any post-translational proteolytic processing in barley, whereas the leaf homologue is processed to a smaller protein. Possible implications of these findings are discussed.

Hysteresis and Reversible Cold-Inactivation of ADP-Glucose Pyrophosphorylase From Barley Seeds

Abstract ADP-glucose pyrophosphorylase (AGP) from barley (Hordeum vulgare L.) seed endosperm showed a lag in activity when assayed after storage at -20 °C. The cold-stored enzyme could regain most, or all, of its activity during 40-60 min following exposure to ambient temperatures. The lags were not observed when 2 mM MgCl2 was added to the storage buffer before freezing. Storage at -20 °C, in the absence of MgCl2, led to the appearance of a low activity A GP form which was activated up to 3-fold by 3-phosphoglycerate (PGA) and had high Km values with ATP of 0.3 and 1.2 mM (with and without PGA, respectively). In contrast, storage at -20 °C in the presence of MgCl2 or incubation at +20 °C resulted in an active enzyme which was only weakly activated by PGA (up to 30%) and had the respective Km values with ATP of 0.1 and 0.3 mM . It is suggested that low temperature may induce a change in the conformation and/or oligomerization state of the AGP protein, resulting in a low activity enzyme form which has distinct regulatory and kinetic properties.