The membrane‐bound inorganic pyrophosphatase (EC 3.6.1.1) from Rhodospirillum rubrum has been investigated with the tools of enzyme kinetics, and with two amino acid reagents, N‐ethyl‐maleimide (MalNET) and 4‐chloro‐7‐nitrobenzofurazan (Nbf‐Cl). The concentration of the true substrate, MgPPi, was varied with constant concentrations of free Mg2+ or PPi. It was observed that Mg2+ acted as an activator. Heat inactivation of the enzyme at 62°C was slowed down in the presence of Mg2+. MalNET and Nbf‐Cl bind to the enzyme, and inhibit its activity. The effect of both reagents is dependent on the temperature. A model is proposed where the 1:1 complex of Mg2+: PPi acts as substrate and Mg2+ interacts directly with the enzyme as an activator. PPi can bind to the enzyme, but is not hydrolyzed in the uncomplexed form.

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A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 11 5E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 11 5E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [ 14C]5'-lp-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgClz or CaC12, whereas, in the presence of MnC1, and ZnC12, the activity was only about 50% of the control. High concentrations of Na2V03 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The K , for ATP and for GTP was 13 pM and 11 pM, respectively, and the V,,, for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.