Immunological Characterization of <i>in Vitro</i> Forms of Homoserine Dehydrogenase from Carrot Suspension Cultures

Turano, Frank J.; Jordan, R.; Matthews, Benjamin F.

doi:10.1104/pp.92.2.395

Cited by 19 publications

(12 citation statements)

References 16 publications

(21 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Identical gels were incubated in the GDH stain solution minus Glu as negative controls. Immunoblot analysis was conducted as described by Turano et al (1990); proteins were resolved in a SDSpolyacrylamide (8.0%) gel (Laemmli, 1970). Rabbit serum raised against grape leaf NAD(H)-GDH was kindly provided by Loulakakis and Roubelakis-Angelakis (1990b).…”

Section: Cel Electrophoresis Cel-staining Procedures and Lmmunoblotmentioning

confidence: 99%

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

et al. 1997

View full text Add to dashboard Cite

Two distinct cDNA clones encoding NAD(H)-dependent glutamate dehydrogenase (NAD[H]-GDH) in Arabidopsis thaliana were identified and sequenced. The genes corresponding to these cDNA clones were designated GDH1 and GDH2. Analysis of the deduced amino acid sequences suggest that both gene products contain putative mitochondrial transit polypeptides and NAD(H)- and α--ketoglutarate-binding domains. Subcellular fractionation confirmed the mitochondrial location of the NAD(H)-GDH isoenzymes. In addition, a putative EF-hand loop, shown to be associated with Ca2+ binding, was identified in the GDH2 gene product but not in the GDH1 gene product. GDH1 encodes a 43.0-kD polypeptide, designated α-, and GDH2 encodes a 42.5-kD polypeptide, designated [beta]. The two subunits combine in different ratios to form seven NAD(H)-GDH isoenzymes. The slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of α- subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of [beta] subunits. GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of α- and [beta] subunits. NAD(H)-GDH isoenzyme patterns varied among different plant organs and in leaves of plants irrigated with different nitrogen sources or subjected to darkness for 4 d. Conversely, there were little or no measurable changes in isoenzyme patterns in roots of plants treated with different nitrogen sources. In most instances, changes in isoenzyme patterns were correlated with relative differences in the level of α- and [beta] subunits. Likewise, the relative difference in the level of α- or [beta] subunits was correlated with changes in the level of GDH1 or GDH2 transcript detected in each sample, suggesting that NAD(H)-GDH activity is controlled at least in part at the transcriptional level.

show abstract

Section: Cel Electrophoresis Cel-staining Procedures and Lmmunoblotmentioning

confidence: 99%

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

et al. 1997

View full text Add to dashboard Cite

show abstract

“…Immunoblotting was conducted as described in Lemoine et al (15), except that the 16-h incubation of the nitrocellulose paper in PBS was omitted and the blocking solution was composed of 5% defatted milk and 0.2% Tween 20 in PBS. Polypeptides were tested for their reactivity with a polyclonal ascitic fluid raised in mouse according to the immunization schedule described by Turano et al (25). The ascitic fluid was raised against the 42-kD polypeptide of PMVs from source leaves previously shown to be involved in the uptake of sucrose (9,15,17).…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Plasma Membrane Vesicles from Source and Sink Leaves

Lemoine¹,

Gallet²,

Gaillard³

et al. 1992

Plant Physiol.

View full text Add to dashboard Cite

Plasma membrane vesicles (PMVs) were prepared by phase partitioning from microsomal fractions of either sink or source leaves of sugar beet (Beta vulgaris L.). The purity, the internal volume, the sidedness, and the sealingness of PMVs prepared from sink leaves did not differ from those measured with PMVs from source leaves. Yet, in response to an imposed proton motive force, PMVs from source leaves accumulated about 4-fold more sucrose than PMVs from sink leaves. The developmental stage did not affect the uptake of glucose and valine in PMVs prepared from leaf tissues. It was concluded that the sink/source transition is accompanied either by the incorporation into the plasma membrane of leaf cells of proteins mediating proton-sucrose cotransport, or by their activation. N-ethylmaleimide and a polyclonal ascitic fluid directed against the 42-kD region of the plasma membrane containing a putative sucrose carrier inhibited the uptake of sucrose in PMVs from source leaves, but not in PMVs from sink leaves. Sodium dodecyl sulfate gel electrophoresis and western blot suggested that the 42 polypeptide was more abundant in the PMVs from source leaves than in the PMVs from sink leaves.

show abstract

“…3). The cross-reactivity with forms of AK by antibody made to purified carrot HSDH (22) was investigated. Equal amounts of protein of each AK form were subjected to PAGE.…”

Section: Resultsmentioning

confidence: 99%

“…Excess protein binding sites were blocked by incubating the filters in 1 x Tris-buffered saline, 1.0% dry milk, and 0.5% BSA for 1 h at room temperature. Nitrocellulose filters were incubated with primary antibody (anti-HSDH antiserum from mouse), secondary and tertiary antibody, and the alkaline phosphatase activity visualized as described (22).…”

Section: Western Blot Analysismentioning

confidence: 99%

See 1 more Smart Citation

Bifunctional Protein in Carrot Contains Both Aspartokinase and Homoserine Dehydrogenase Activities

Wilson

Gray²,

Matthews³

1991

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia co/i, which has two bifunctional enzymes, aspartokinase 1-homoserine dehydrogenase I and aspartokinase 11-homoserine dehydrogenase I. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coii ThrA gene product aspartokinase 1-homoserine dehydrogenase 1.

show abstract

Immunological Characterization of in Vitro Forms of Homoserine Dehydrogenase from Carrot Suspension Cultures

Cited by 19 publications

References 16 publications

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

Plasma Membrane Vesicles from Source and Sink Leaves

Bifunctional Protein in Carrot Contains Both Aspartokinase and Homoserine Dehydrogenase Activities

Contact Info

Product

Resources

About