RNA 5′-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5′-triphosphatase that hydrolyzes the γ phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and P_i (K_m = 43 μM ATP; V_max = 30 s⁻¹) and GTP to GDP and P_i. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.     

5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme

5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn²⁺ or Mg²⁺, and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH₄⁺ or K⁺. The K_m for ATP is 0.4 mm and for 5-oxo-l-proline is 14 μm. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a γ-glutamyl cycle in higher plants.     

A comparative study of the protein components of ribonucleoprotein particles isolated from rat liver and hepatoma nuclei

To solve the mechanism for the complete cessation of DNA synthesis in Tetrahymena cells involved in the amino acid starvation, the nature of DNA polymerase activity was investigated in crude enzyme preparations or in toluene-permeabilized specimens. In crude enzyme preparations from growing cells, ³H-TTP incorporation into acid-insoluble products showed little dependency on exogenous DNA template, while incorporation increased markedly in the presence of ATP. These characteristics were very similar to those of replicative DNA synthesis in permeabilized Escherichia coli.Variations of DNA and RNA polymerase activities following transfer of exponentially growing Tetrahymena cells to amino acid-deprived medium showed that in the crude enzyme preparations DNA polymerase activity dropped sharply within 3 h after the transfer and practically no activity was detected thereafter, whereas RNA polymerase activity did not disappear in the same preparations. Such enzyme kinetics coincided well with the kinetics of in vivo synthesis of the corresponding nucleic acid.The cessation of DNA synthesis in the amino acid-starved cells may be due not to the activation of DNase or a soluble polymerase inhibitor, nor to the deficiency of each kind of deoxyribonucleoside triphosphate or magnesium ion or ATP generation system. It follows from this that the cessation of DNA polymerase activity in the starved cells may be due to the deficiency of DNA polymerase or its associated factor(s) as a reflection of short life-span of such a protein.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

Lysophosphatidylcholine-activated,vanadate-inhibited,Mg2+-ATPase from radish microsomes

An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg²⁺-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol P_i · min^?1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg²⁺ or some other divalent cations (CO²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Zn²⁺ in order of decreasing effectiveness); Ca²⁺ could not substitute for Mg²⁺ and was strongly inhibitory in its presence. K⁺, Rb⁺ and Na⁺ and also to a lesser extent NH₄⁺ and Li⁺ were significantly stimulatory, while the anions NO₃^?, H₂PO₄^?, Cl^? and SO₄^2? were ineffective. Orthovanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the K_m for ATP with that for Mg²⁺ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent K_m of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.     

Inhibition of Leukaemia Virus Replication by Polyadenylic Acid

A PREVIOUS communication from this laboratory demonstrated that the DNA polymerase of the Rauscher leukaemia virus is strongly inhibited in vitro by unprimed, single stranded polyribonucleotides¹ as a result of competition between the polymers and the active template for the same enzyme binding site. This inhibition was apparently specific, since partially purified preparations of DNA polymerase from Escherichia coli and BALB/c mouse embryos were not inhibited in the same conditions. We attempted to determine therefore whether single stranded polyribonucleotides would have any effect on the activities of oncogenic RNA viruses in cultured cells.     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

Properties of CAR-kinase: The enzyme that phosphorylates the cAMP chemotactic receptor ofD. discoideum

The cell surface cAMP chemotactic receptor ofD. discoideum can be phosphorylated in partially purified plasma membrane preparations in a ligand-dependent manner. CAR-kinase, the enzyme responsible for receptor phosphorylation, was shown to be an integral membrane protein. It could utilize either ATP or GTP to phosphorylate the receptor, although ATP was much more efficient. The apparent affinity constant for ATP was approximately 20–25 µM. Maximum CAR-kinase activity was observed betweenpH 6.5 andpH 7, and required the presence of Mg²⁺. Neither Mn²⁺ nor Ca²⁺ could substitute for that divalent cation. The enzyme was found to be sensitive to the ionic strength and temperature of the incubation reaction. Dephosphorylation of the receptor was not observed in the membrane preparations, indicating that the enhanced level of receptor phosphorylation that occurred upon ligand binding was not an indirect reflection of receptor dephosphorylation and subsequent incorporation of radiolabeled phosphate.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468–7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833–2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of M_r 42000 and 25000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.     

A simple and defined method to study calcification by isolated matrix vesicles effect of ATP and vesicle phosphatase

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 μg of protein, were incubated for 2 h in a ⁴⁵Ca-labelled solution with 2.2 mM Ca²⁺, 1.6 mM PO₄^3? and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO₄ hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca²⁺ or Mg²⁺ was found to stimulate matrix vesicle. ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, β-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, veiscles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Purification and Characterization of Phosphoribulokinase from Immature Pods of Brassica

Phosphoribulokinase (ATP:D — ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19; PRuK) from immature pods of Brassica was purified to apparent homogeneity with about 31% recovery using ammonium sulphate fractionation, gel filtration through Sepharose CL-6B and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme, having molecular mass of about 180 kD, was heterotetramer with subunit molecular mass of 48, 47, 41 and 33 kD. The enzyme had an absolute requirement for a divalent cation Mg²⁺ and a monovalent cation K⁺for optimal activity. At optimum pH of 8.0–8.4, the enzyme showed typical hyperbolic response for both the substrates with Km values of 333 μM and 100 μM, respectively for Ru5P and ATP. The enzyme was inhibited by RU-1, 5-P₂, 6-phosphogluconate and AMP, and activatded by glu-1-P, glu-6-P and Pl. RU-1, 5-P₂ and 6-phosphogluconate inhibited the enzyme competitively with respect to Ru5P and non-competitively with respect to ATP. It appears that the activity of the Brassica pod enzyme besides being controlled at the level of metabolites, is regulated by light and energy status of the cell.