R.C. Sachar

Induction of acid phosphatase in cotton seedlings: Enzyme purification, subunit structure and kinetic properties

Abstract About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of 35 S from 35 SO 2− 4 into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M , of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K m of the enzyme with p -nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p -nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.

Regulation of poly(A) polymerase activity and poly(A)+ RNA levels by auxin in pea epicotyls

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Phosphorylation of rubisco in Cicer rietinum: Non-phosphoprotein nature of rubisco in Nicotiana tabacun

Abstract Leaf discs from Cicer and Nicotiana were incubated in a solution of [ 32 P]-orthophosphate (24 hr). RUBISCO was purified from these and electrophoresed on native PAGE. A single protein stained band was observed on gels, thus proving the electrophoretic homogeneity of RUBISCO both in Cicer and Nicotiana . Autoradiography of gels revealed a radioactive band in the zone of protein stained band of Cicer RUBISCO, whereas no radioactivity was detected in the region of the protein stained band of Nicotiana RUBISCO. This suggested that Cicer RUBISCO unlike that of Nicotiana is a phosphoprotein. Further analysis of [ 32 P]-labelled purified Cicer RUBISCO on SDS—PAGE showed two protein stained bands corresponding to the large and small subunits of the enzyme. Autoradiography of the denaturing gel showed a radioactive band exclusively in the zone of the small subunit. Chemical characterization of [ 32 P]-labelled Cicer RUBISCO by acid hydrolysis showed radioactivity in phosphoserine and phosphotyrosine. Treatment of [ 32 P]-labelled Cicer RUBISCO with purified alkaline phosphatase brought about a significant dephosphorylation (∼70%) of the enzyme. The in vitro dephosphorylation of Cicer RUBISCO resulted in the dissociation of the small subunits from the catalytic octameric large subunits. Concomitantly, there was a marked decrease in the catalytic activity of the dephosphorylated Cicer RUBISCO in comparison with the phosphorylated form of RUBISCO. Phosphorylation of the small subunit seems crucial for the assembly of large and small subunits and thus contributes to the regulation of Cicer RUBISCO. By contrast, Nicotiana RUBISCO showed no loss of catalytic activity following its treatment with alkaline phosphatase.

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS‐polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [32P]orthophosphate. Autoradiographic analysis of 32P‐labelled RuBPCase on SDS‐PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls

A significant stimulation (2‐ to 2.5‐fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA3, 1μM)‐treated epicotyls of the dwarf pea (Pisum sativum). This was accompanied by a 2.4‐fold increase in the endogenous pool of S‐adenosylmethionine. Both abscisic acid (10 μM) and cycloheximide (20 ) inhibited the GA3‐mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA3‐treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA3 treatment. High concentration of L‐methionine (2 mM) mimicked the GA3‐elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.

Differential regulation of S-adenosylmethionine synthetase isozymes by gibberellic acid in dwarf pea epicotyls

Dwarf pea epicotyls contained a single activity peak of S-adenosylmethionine (AdoMet) synthetase (isozyme I). Gibberellic acid (GA3, 1 microM) induced two additional isozymes (II and III). Cycloheximide (20 microgram/ml) blocked the appearance of GA3-induced isozymes, suggesting that it is dependent on de novo protein synthesis. Conclusive proof was obtained by labelling the isozymes II and III with [35S]SO2-(4) in vivo. The purified 35S-labelled AdoMet synthetase isozymes (II, III) showed a single protein band that coincided with the single radioactive peak on SDS-PAGE. Molecular-sieve chromatography of the isozyme I from control dwarf pea epicotyls and three isozymes of GA3-treated epicotyls on Sepharose CL-6B showed a single activity peak with an identical molecular mass of 174 kDa for each isozyme. Analysis of purified AdoMet synthetase isozymes (I, II, III) on SDS-PAGE showed a single silver-stained protein band with a molecular mass of 87 kDa. This proved the dimeric nature of all the isozymes of AdoMet synthetase which could be physically separated by ion-exchange chromatography on DE-52. In vitro molecular hybridization of physically separated isozymes by NaCl-freeze-thaw treatment method revealed that the three isozymes (I, II, III) in GA3-treated dwarf pea epicotyls are formed through the random dimerization of two different species of enzyme subunits that differ in their net charge. Thus, the two flanking activity peaks (isozymes I, III) represent homodimers, while the middle activity peak (isozyme II) is a heterodimer. Apparently, the single isozyme I in control epicotyls is a product of one gene of AdoMet synthetase (SAM 1), while three isozymes in GA3-treated epicotyls are the product of two genes of AdoMet synthetase. We speculate that the differential regulation of AdoMet synthetase in GA3-treated epicotyls is achieved by the expression of an alternate gene of AdoMet synthetase (SAM 2).

Purification and characterization of poly(A) polymerase from germinated wheat embryos: enzyme glycosylation

Abstract Wheat embryo poly(A) polymerase was purified (1348-fold) to electrophoretic homogeneity by adenosine triphosphate(ATP)-Sepharose and concanavalin A(Con A)-agarose affinity chromatography. The purified polymerase was devoid of cryptic nuclease activity after Con A-agarose affinity chromatography. Thus the Con A-agarose fraction showed a linear increase in poly(A) polymerase activity as a function of time up to 90 min. Fractionation of purified enzyme on native PAGE showed a single protein stained band that coincided with the activity peak of poly(A) polymerase. The molecular weight of poly(A) polymerase was 65 000–70 000 as determined by molecular sieve chromatography. A single subunit of purified poly(A) polymerase (mol. wt., 64 000) was observed on SDS-PAGE. This proved the monomeric nature of the enzyme. The binding of poly(A) polymerase to Con A-agarose and its elution with α-methyl mannopyranoside suggested its glycoprotein nature. The apparent Km of poly(A) polymerase for ATP was 86 μM. The 3H-labelled reaction product of purified enzyme binds to oligo(dT)-cellulose affinity matrix. In addition, the putative poly(A) product was not hydrolysed by RNase A and RNase Ti. Thus, it was proved that poly(A) polymerase catalyzes the synthesis of poly(A) sequences.

De novo synthesis of poly(A) polymerase in mung bean hypocotyls, involving stored mRNA

Abstract A linear increase in poly(A) polymerase activity (3–4-fold) in hypocotyls of germinating mung beans was accompanied by a parallel increase in the levels of poly(A) + RNA (4–16 hr). The enzyme activity declined at subsequent stages of seed germination (28–52 hr) without a similar downward trend in the levels of poly(A) + RNA. The inhibition of enzyme activity could, however, be alleviated by ion exchange chromatography on DE-52. This indicated the presence of some inhibitory factor which interfered with the poly(A) polymerase activity in the ammonium sulphate fraction precipitate. Thus a direct relationship is indicated between the rise in the activity of poly(A) polymerase and the increased levels of poly(A) + RNA in the hypocotyls of germinating embryos. Administration of cycloheximide (20 μg/ml) strongly inhibited poly(A) polymerase activity in the hypocotyls, thereby indicating the requirement of de novo protein synthesis. Conclusive proof for the de novo synthesis of poly(A) polymerase was achieved by labelling the enzyme in vivo with 35 SO 4 2- . The 35 S-label was recovered in the cysteine and methionine residues of the purified poly(A) polymerase. Thus it became evident that the stimulation of poly(A) polymerase activity in hypocotyls is primarily achieved by the de novo synthesis of the enzyme. Transcriptional activity was, however, not mandatory for the synthesis of poly(A) polymerase, since cordycepin (250 μM) failed to block the rise in the enzyme activity. Apparently, poly(A) polymerase is synthesized from its stored mRNA in mung bean hypocotyls. Curiously, the cordycepin-treated hypocotyls exhibited a significant stimulation of poly(A) polymerase activity (3–4 fold) over that of the controls. It is envisaged that the drug blocks the transcription of the inhibitory factor of poly(A) polymerase and is therefore indirectly responsible for the stimulation of enzyme activity. The 3 H-labelled reaction product of poly(A) polymerase was resistant to the hydrolytic action of RNAase A and RNAase T 1 . The labelled product was also retained on the affinity matrix of oligo (dT)-cellulose. This proved the polyadenylate nature of the in vitro synthesized reaction product. The M r of the mung bean poly(A) polymerase is 120000.