Separation in Solution

Scopes, Robert K.

doi:10.1007/978-1-4757-1770-9_5

Cited by 2 publications

(1 citation statement)

References 2 publications

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“…In contrast, the mo1 wt of the native ICDH2 holoenzyme is compatible with a protein of two or three identical subunits. However, when using a gel-filtration column, the elution volume from which the apparent mo1 wt is calculated is theoretically related to the Stokes radius, not to size or mo1 wt (Scopes, 1982). Consequently, if the shapes of the unknown and standard proteins are dissimilar, the determination of the apparent mo1 wt from the elution volume can be in error.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

et al. 1994

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Creen, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes. lheir elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves. lCDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process. Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anionexchange and affinity chromatography. l h e isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833-and 1328-fold for ICDH1 and ICDH2, respectively. Polyclonal antibodies were raised against each isoform. The ICDHZspecific antibody was used to localize tobacco leaf lCDH2 in situ by an immunogold labeling technique. The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves. ICDHl and lCDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits. Similar apparent K,,, values for NADP, D(+)isocitrate, and Mgz+ were found for the two enzymes when assayed with Mgz+ as the metal cofactor.

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Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

et al. 1994

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Isolation of Mucor Miehei and M. Pusillus Aspartic Proteinases From Partially Purified Sources Using Preparative Isoelectric Focusing

Wynne

Yada

1991

J Food Biochemistry

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Dalysis, preparative isoelectric fbcusing ( I E q and gel jltration were employed to obtain Jinal enrichments of 2.2-fbld and 1.4fbldfbr Mucor miehei (CBS 360.75) proteinase (MMP) and M. pusillus (var. Lind) proteinase (MPP) , respectively, from partially purijed sources. Recovery of total activity (percent yield of MMP was 38% fbllowing preparative IEF and 6.7% ajer gel jltration. Percent yield of MPP was 61% fbllowing preparative IEF and 15% ajer gel jltration. Preparative IEFfractionation with a low pH (3-5) ampholyre produced most of the increase, and partitioned b r o w pigments into fractions of p H 2.0 to 3.5. 7he protocol produced proteinases of high purity as indicated by SDS PAGE. 7he isoelectric point, molecular weight, eainction coeflcient and amino acid composition of MMP and MPP proteinases were in agreement with previously determined values. Secondary structure analysis conjnnedpreviousjndings that these tnzymes contain a high proportion of @sheet structure. Pun3cations starting with 4 to 5 g of crude partiallypurijed MMPpreparation (at least 50% NaCl) yielded between 18 and 28 mg of purijed protein.lCorrespondence to: Ms.

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Separation in Solution

Cited by 2 publications

References 2 publications

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme)

Isolation of Mucor Miehei and M. Pusillus Aspartic Proteinases From Partially Purified Sources Using Preparative Isoelectric Focusing

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