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We have isolated a cDNA clone, designated ZLKRSDH , encoding the bifunctional enzyme lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) from maize. The predicted polypeptide has an N-terminal LKR domain and a C-terminal SDH domain that are similar to the yeast LYS1 and LYS9 monofunctional proteins, respectively. The maize LKR/SDH protein is located in the cytoplasm of subaleurone endosperm cell layers. Transcripts and polypeptides as well as enzyme activities showed an upregulation and downregulation during endosperm development. The developmental expression of ZLKRSDH was examined in normal and opaque2 seeds. In the mutant endosperm, mRNA levels were reduced by Ͼ 90%, with concomitant reductions in polypeptide levels and LKR/SDH activity. These results suggest that lysine levels in the endosperm are likely to be controlled at the transcriptional level by the Opaque2 transcription factor. INTRODUCTIONSince the discovery of the high-lysine opaque2 ( o2 ) maize mutant (Mertz et al., 1964), research efforts have been directed toward understanding the biochemical and molecular mechanisms leading to the increase in lysine content in the endosperm. Studies conducted during the past 30 years revealed that the homozygous o2 mutation causes an ‫ف‬ 70% reduction in zein content and affects the content of a number of proteins and enzymes related to nitrogen and sugar metabolism in the maize endosperm (Habben et al., 1993; Giroux et al., 1994; Gallusci et al., 1996). The cloning of the O2 gene revealed that it encodes a basic leucine zipper protein transcription factor involved in the transcriptional control of the zein genes, the b-32 ribosome-inactivating protein, and cytoplasmic pyruvate orthophosphate dikinase (cyPPDK), an enzyme involved in carbon partitioning (Lohmer et al., 1991; Bass et al., 1992;Schmidt et al., 1992; Cord Neto et al., 1995; Gallusci et al., 1996). Lysine catabolism also is affected in the o2 mutant. 14 C-lysine feeding experiments revealed that lysine was converted to glutamic acid and proline but to a much lesser extent in o2 than in normal endosperm (Sodek and Wilson, 1970).Despite the observation of extensive lysine degradation in maize seeds (Sodek and Wilson, 1970;da Silva and Arruda, 1979), there have been several attempts to establish positive selection strategies to isolate mutants with aspartate kinase (AK) insensitivity to feedback inhibition caused by lysine. AK is the first regulatory enzyme of the aspartate pathway, which leads to the biosynthesis of lysine, methionine, threonine, and isoleucine. Thus, the derepression of AK would result in an overproduction of these amino acids (reviewed in Azevedo et al., 1997). AK-insensitive mutants that overproduce threonine have been described in barley (Bright et al., 1982), maize (Diedrick et al., 1990), and tobacco (Frankard et al., 1992), but they exhibit only marginal effects on lysine accumulation. The absence of lysine overproduction in these mutants was attributed to the second regulatory enzyme, dihydrodipicolinate syn...

A cDNA clone (AtPUMP) encoding a plant uncoupling mitochondrial protein was isolated from Arabidopsis thaliana. The cDNA contains an open reading frame of 921 nucleotides encoding 306 amino acids (predicted molecular weight 32 708). The predicted polypeptide is 81% identical and 89% similar to the potato UCP-like protein, and includes an energy transfer protein motif common to mitochondrial transporters. The AtPUMP gene exists as a single copy in the Arabidopsis genome. The corresponding transcript was expressed in all tissues and was strongly induced by cold treatment. We suggest that the putative AtPUMP protein may play a role in heat-requiring physiological events in Arabidopsis.z 1998 Federation of European Biochemical Societies.

Insulin and leptin have overlapping effects in the control of energy homeostasis, but the molecular basis of this synergism is unknown. Insulin signals through a receptor tyrosine kinase that phosphorylates and activates the docking proteins IRSs (insulin receptor substrates), whereas the leptin receptor and its associated protein tyrosine kinase JAK2 (Janus kinase 2) mediate phosphorylation and activation of the transcription factor STAT3 (signal transducer and activator of transcription). Here, we present evidence for the integration of leptin and insulin signals in the hypothalamus. Insulin induced JAK2 tyrosine phosphorylation, leptin receptor phosphorylation which, in the presence of leptin, augmented the interaction between STAT3 and this receptor. Insulin also increased the leptin-induced phosphorylation of STAT3 and its activation. These results indicate that insulin modulates the leptin signal transduction pathway, and may provide a molecular basis for the coordinated effects of insulin and leptin in feeding behavior and weight control. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

l h e first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and a-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to a-aminoadipic-6-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. l h e reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. l h e highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysineketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide.

Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in β-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2α phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in β-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in β-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and a-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.There is little information on lysine catabolism in higher plants. Most of the available data were obtained in studies on the incorporation and metabolism of radiolabeled precursors by plant tissues. Feeding experiments with ["4C]lysine demonstrated the incorporation of radioactivity into a-amino adipic acid and glutamic acid in wheat (18) and into saccharopine and diaminopimelic acid in maize and barley (16,26). In developing endosperm of maize and barley, radiolabeled lysine is incorporated primarily into glutamic acid and proline (4, 26). These findings indicate that lysine is catabolized in plants via the saccharopine pathway.The first enzymatic evidence for the operation of the saccharopine pathway for lysine catabolism in plants was obtained with the demonstration of LKR3 activity in immature endosperm of maize (3). LKR (EC 1.5.1.8) condenses lysine and a-ketoglutarate into saccharopine using NADPH as cofactor.An understanding of the pathways for lysine biosynthesis and degradation in plants has enormous importance because of the limiting concentration of this essential amino acid in major food sources such as cereals. Valuable information can be obtained by the elucidation of the properties of enzymes involved in the biosynthesis and catabolism of lysine and by the use of mutants in which the activities of the enzymes are altered.Since the discovery of the superior nutritive value of the high lysine maize mutant opaque-2 (15), there have been many studies on the effects of this mutant gene on protein and amino acid metabolism in maize endosperm. The opaque-2 gene is located on the short arm of chromosome 7 and its major effect is the reduction of the maize storage protein zein. This is a complex of polypeptides coded by a multigenic family located on chromosomes 4 and 7 (23). The opaque-2 gene in the homozygous form reduces the zein content of the endosperm by up to 70% (6). The reduction is

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (α') subunit of β-conglycinin tissue-specific promoter from soybean and the α-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels of bioactive hGH up to 2.9% of the total soluble seed protein content (corresponding to approximately 9 g kg(-1)) were measured in mature dry soybean seeds. The results of ultrastructural immunocytochemistry assays indicated that the recombinant hGH in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Specific bioassays demonstrated that the hGH expressed in the soybean seeds was fully active. The recombinant hGH protein sequence was confirmed by mass spectrometry characterisation. These results demonstrate that the utilisation of tissue-specific regulatory sequences is an attractive and viable option for achieving high-yield production of recombinant proteins in stable transgenic soybean seeds.