Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures

Yeung, Anthony T.; Turner, Katherine J.; Bascomb, Newell F.; Schmidt, Robert R.

doi:10.1016/0003-2697(81)90138-x

Cited by 23 publications

(49 citation statements)

References 17 publications

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“…Crucial to the success of this procedure is the ability of antibodies, prepared against the native enzyme or its subunits, to recognize the growing nascent polypeptide chains on intact polysomes engaged in synthesis of the enzyme. Earlier in this laboratory (5,27), antibodies were prepared in rabbits against the purified NADP-GDH holoenzyme. The anti-NADP-GDH IgG was purified by antigen-affinity chromatography and shown to be monospecific by a number ofimmunochemical procedures (27).…”

Section: Resultsmentioning

confidence: 99%

“…Earlier in this laboratory (5,27), antibodies were prepared in rabbits against the purified NADP-GDH holoenzyme. The anti-NADP-GDH IgG was purified by antigen-affinity chromatography and shown to be monospecific by a number ofimmunochemical procedures (27). Moreover, the affinity-purified anti-NADP-GDH IgG was labeled with 1251, and used in binding studies with intact total cellular polysomes isolated from ammonium-induced cells of C. sorokiniana (23).…”

Section: Resultsmentioning

confidence: 99%

“…After 5 to 10 min, the anti-antibody cellulose was removed from the mixture by centrifugation for 5 (23). To estimate the amount ofcontamination ofthe immunoselected NADP-GDH mRNA by other translatable mRNAs, the in vitro translation of the immunoselected poly(A)+RNA was allowed to proceed for 60 min, and the radioactive proteins that were synthesized in vitro were subjected to SDS-PAGE as described earlier (23,27). The gels were fixed, dried, and autoradiograms prepared.…”

Section: Methodsmentioning

confidence: 99%

“…Recent research has been focused on the expression of the gene(s) coding for a chloroplast-localized (15) NADP-GDH4 that is inducible by ammonium in Chlorella sorokiniana cells (12,20). By use of immunochemical and biochemical procedures (27), the NADP-GDH has been shown to be synthesized as a precursor protein on cytosolic polysomes (15) and rapid degradation in both uninduced and induced cells (1,23), and to undergo rapid inactivation after removal of ammonium from fully-induced cells (1). Because of these post-translational processes, the catalytic activity or amount of antigen of this enzyme cannot be used as a reliable measure of the level of expression of its structural gene in cell cycle experiments (7,16,20,21,26) nor in experiments in which changes are made in the nitrogen nutritional status of the cells (1,6).…”

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confidence: 99%

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Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Bascomb¹,

Turner²,

Schmidt³

1986

Plant Physiol.

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A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)+RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)'RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Bascomb¹,

Turner²,

Schmidt³

1986

Plant Physiol.

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“…First, from a cellular energetics standpoint, it seemed unlikely that an enzyme, such as RuBPCase, representing 10% of the total cellular soluble protein (17), would undergo rapid synthesis and degradation throughout the Chlorella cell cycle. Second, by use of immunochemical procedures (21), cycloheximide was shown (2,12) to affect the rate of degradation of another enzyme in C. soroki-niana, and also to affect processes other than protein synthesis in other organisms (1,6,7,11,16). Third, by use of nonimmunochemical procedures, Iwaij et al (13) were unable to demonstrate turnover of RuBPCase antigen during the light-phase of the cell cycle of Chlamydomonas.…”

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confidence: 99%

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

Toman

Schmidt²

1985

Plant Physiol.

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ABSTRACIthen remained constant for several hours before increasing once again at the beginning ofthe next cell cycle (i.e. 10th h; midpoint ofthe period ofcell division). This step pattern could be changed to a continuous pattern of accumulation of carboxylase activity merely by increasing the light intensity to 1 100 ft-c and as a result doubling the growth rate ofthe synchronous cells. Because the increase in carboxylase activity could be blocked by cycloheximide (2.5 isg/ml culture), the increase in enzyme activity during the cell cycle was assumed to be due to de novo synthesis of the carboxylase. These aforementioned observations taken collectively were interpreted as indicating that the structural genes of this enzyme might be continuously available for transcription with their expression regulated by oscillatory repression (4, 8) as proposed for bacteria.In a later study, Sitz et al. (15) used actinomycin D to show that the cell cycle accumulation of RuBPCase activity in this organism was also dependent upon RNA synthesis. These workers also attempted to determine if changes in the rate of enzyme turnover (i.e. degradation) might be affecting the pattern of accumulation of the enzyme during the cell cycle. They showed that the concentration of cycloheximide (2.5 gg/ml) used by Molloy and Schmidt (14) was insufficient to inhibit total cellular protein synthesis. When the concentration of this inhibitor was increased to 12 ,g/ml of culture or greater, total cellular prqtein ceased to accumulate and the activity ofthe carboxylase decayed at a faster rate (t½ = 4.4 h) than the rate (t½h = 9 h) observed in the earlier study (14). In fact, because the enzyme decayed with the same half-life of 4.4 h at all times tested during the cell cycle, including the period of constant enzyme activity, it was concluded that enzyme synthesis and degradation occurred throughout the cell cycle ofChlorella. During periods ofconstant enzyme activity, a steady state was assumed to exist between enzyme synthesis and degradation. Thus, the structural genes encoding the subunits of RuBPCase appeared to be expressed throughout the cell cycle with changes in the rate of carboxylase synthesis rather than in the rate of degradation determining the time of accumulation of enzyme activity during the cell cycle.Several considerations made the conclusions ofthe aforementioned cell cycle studies difficult to accept. First, from a cellular energetics standpoint, it seemed unlikely that an enzyme, such as RuBPCase, representing 10% of the total cellular soluble protein (17)

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Affinity Chromatography

Lowe

Clonis

1985

Bioactive Polymeric Systems

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Two types of resin-conjugated Tamiflu analogs were synthesized by modifying our original synthetic route of oseltamivir phosphate (Tamiflu). The prepared resins bound to influenza virus neuraminidase, the main target of Tamiflu. The resins will be useful for isolating and identifying presumed endogenous vertebrate proteins that interact with Tamiflu, which might relate to the rarely observed abnormal behavior exhibited by some influenza patients treated with Tamiflu.

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Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures

Cited by 23 publications

References 17 publications

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

Affinity Chromatography

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