| Summary: | Haemophilus influenzae, as well as other members of the genus which require V-factor, display a unique growth requirement for intact NAD. This organism, the primary cause of bacterial meningitis, is incapable of synthesis of pyridine nucleotides from the usual precursors.
An externally directed nucleotide pyrophosphatase was extracted from the organism and purified 700-fold using ammonium sulfate precipitation, ion-exchange and affinity chromatography. The enzyme was determined to be a periplasmic glycoprotein consisting of a single polypeptide of M<sub>r</sub>= 65,000. The enzyme had a pH optimum over the range .pH 8.0-9.0 and was not activated by the addition of mono or divalent cations, nor was it inhibited by EDTA. The enzyme was observed to have a broad substrate specificity and functioned in a manner indicative of negative cooperativity with all substrates except several modified in the adenine ring. The most effective inducer of negative cooperativity was NAD as indicated by its Hill coefficient of 0.26. The enzyme was inhibited by adenine nucleotides _ and 5'-AMP, at 20 μM, abolished the negative cooperativity of the enzyme. The enzyme was determined to possess excitation and emission maxima at 286 and 337 nm, respectively, indicative of the presence of tryptophan. The fluorescence of the enzyme was quenched by addition of aliquots of adenine nucleotides. The quenching occurred in a biphasic manner. The enzyme was inactivated by 2,3- butanedione and by Woodward's Reagent K.
Studies of the ability of compounds to serve as V-factor revealed that nicotinamide mononucleotide (NM), NAD, as well as analogs of NAD, served as V-factor. The ability of compounds to inhibit growth was also accessed, and the growth of the organism was seen to be inhibited by adenine nucleotides as well as other compounds. The inhibition of growth of Haemophilus influenzae has important clinical implications which are discussed, as well as a model of the NAD metabolism of the organism which is presented. === Ph. D.
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