vvp5cs  (Bio-Rad)

Journal:

Article Title: Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of ? 1 -Pyrroline-5-Carboxylate Synthetase mRNA or Protein 1

doi:

Figure Lengend Snippet: V. vinifera P5CS shares a high degree of homology with other P5CS sequences. Multiple alignment of predicted VVP5CS protein sequence with deduced amino acid sequences obtained from nucleotide databases with the following accession numbers: V. vinifera (accession no. AJ005686) from this study, Arabidopsis (A. thaliana, accession no. AT-P5CS1; SwissProt no. P54887), V. aconitifolia (accession no. P32296), and E. coli ProA and ProB (accession nos. P07004 and P07005, respectively). Residues identical to the V. vinifera sequence are highlighted. Amino acids involved in Pro feedback inhibition of the V. aconitifolia P5CS enzyme are indicated with asterisks (Zhang et al., 1995). Degenerate oligonucleotides used in reverse-transcription-PCR were designed based on the sequences that are underlined.

Article Snippet: Recombinant VVP5CS was purified using Talon Metal Affinity Resin (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions, except that protein was eluted in buffers containing 20% (v/v) glycerol.

Techniques: Sequencing, Inhibition

Journal:

Article Title: Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of ? 1 -Pyrroline-5-Carboxylate Synthetase mRNA or Protein 1

doi:

Figure Lengend Snippet: Pro feedback inhibition of VVP5CS expressed in E. coli and purified by affinity chromatography. P5CS assays were conducted in the presence of the Pro and Glu concentrations indicated. Results are expressed as the percentages of specific activity in the absence of Pro.

Article Snippet: Recombinant VVP5CS was purified using Talon Metal Affinity Resin (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions, except that protein was eluted in buffers containing 20% (v/v) glycerol.

Techniques: Inhibition, Purification, Affinity Chromatography, Activity Assay

Journal:

Article Title: Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of ? 1 -Pyrroline-5-Carboxylate Synthetase mRNA or Protein 1

doi:

Figure Lengend Snippet: Southern analysis of grapevine genomic DNA indicates that VVP5CS is encoded by a single gene. DNA isolated from V. vinifera cv Chardonnay was digested with the restriction enzymes PstI, XbaI, HindIII, and StyI, probed with a 1.1-kb fragment of VVP5CS cDNA (nucleotides 590–1701), and then screened under high- and low-stringency conditions. Since both sets of conditions produced identical results, only those obtained after the high-stringency screen are shown.

Article Snippet: Recombinant VVP5CS was purified using Talon Metal Affinity Resin (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions, except that protein was eluted in buffers containing 20% (v/v) glycerol.

Techniques: Isolation, Produced

Journal:

Article Title: Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of ? 1 -Pyrroline-5-Carboxylate Synthetase mRNA or Protein 1

doi:

Figure Lengend Snippet: Steady-state levels of VVP5CS mRNA throughout fruit development. A, Total RNA (15 μg) isolated from V. vinifera cv Chardonnay berries was electrophoresed, blotted onto a nylon membrane, and probed with a 0.89-kb fragment of VVP5CS cDNA (nucleotides 791–1680). B, A replica gel was stained with ethidium bromide to demonstrate the equivalence of RNA loading in each lane. C, Relative levels of VVP5CS transcript detected.

Article Snippet: Recombinant VVP5CS was purified using Talon Metal Affinity Resin (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions, except that protein was eluted in buffers containing 20% (v/v) glycerol.

Techniques: Isolation, Staining

Journal:

Article Title: Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of ? 1 -Pyrroline-5-Carboxylate Synthetase mRNA or Protein 1

doi:

Figure Lengend Snippet: Steady-state levels of VVP5CS and PDH proteins throughout fruit development. A, Protein extracts from whole-berry homogenates of V. vinifera cv Chardonnay were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Each lane contained extracts from equivalent amounts of berry homogenate on a fresh weight basis. B and D, Replica gels were transferred to a nitrocellulose membrane and subjected to western analysis using antibodies prepared against VVP5CS (B) or AtPDH (D). C and E, Relative levels of VVP5CS and PDH proteins.

Article Snippet: Recombinant VVP5CS was purified using Talon Metal Affinity Resin (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions, except that protein was eluted in buffers containing 20% (v/v) glycerol.

Techniques: SDS Page, Staining, Western Blot