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The N-terminal 15 amino acids of the major protein associated with inorganic pyrophosphatase activity in Bacillus subtilis WB600 are identical to those of B. subtilis ORF yybQ. This ORF was amplified from B. subtilis WB600 DNA by PCR and cloned into an overexpression vector in Escherichia coli. Induction of overexpression produced a soluble protein of 34000 Da by SDS-PAGE and by matrix-assisted laser desorption and ionization mass spectrometry. The overexpressed protein had a high specific activity for the hydrolysis of magnesium pyrophosphate, and was specifically and reversibly activated by Mn2+ ions. These properties are identical to those of inorganic pyrophosphatase purified from B. subtilis WB600. No significant similarity was found between the derived sequence of the B. subtilis yybQ-encoded protein and published sequences of identified inorganic pyrophosphatases of Eukarya, Bacteria or Archaea domains. However, there is significant similarity to three putative proteins of unknown function from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from Streptococcus gordonii. The genomes of B. subtilis, M. jannaschii and A. fulgidus do not contain sequences similar to those of hitherto known soluble inorganic pyrophosphatases. The present findings, together with a survey of the properties of inorganic pyrophosphatases from 38 different sources, suggest that the B. subtilis yybQencoded protein is the first fully characterized member of a new class of inorganic pyrop hosp hatase.I

1. UDP-galactose utilization by isolated Golgi vesicles or rat mammary gland synthesizing lactose causes accumulation of UMP but not UDP, although UDP is the immediate product of lactose synthase (EC 2.4.1.22). 2. This can be ascribed to a nucleoside diphosphatase (EC 3.6.1.6), specific for UDP, GDP and IDP, activated by bivalent metal ions and apparently located on the luminal face of the Golgi membrane. 3. The uridine diphosphatase activity exceeds the total galactosyltransferase activity 5-fold, and is estimated to maintain UDP at about 14 micrometer within the Golgi lumen. 4. Evidence is given that UMP, but not UDP, penetrates the membrane and that UMP is rephosphorylated to UDP by a UMP kinase located in the cytosol. 5. Golgi-cytosol relationships with respect to lactose synthesis are formulated in terms of a uridine nucleotide cycle which throws new light on the energy cost and possible regulation of lactose synthesis.

Elucidation of the details of lactose synthesis, in particular its dependence upon alpha-lactalbumin and its location within the lumen of the Golgi apparatus, now allows one to ask useful questions pertaining to its regulation. Attention is directed towards galactosyltransferase itself (EC 2.4.1.22), which appears to be rate-limiting in the uridine nucleotide cycle that supports lactose synthesis, and to those factors that may affect its activity. In laboratory animals alpha-lactalbumin appears to be the major agent of regulation during lactogenesis but is not necessarily limiting at other times, whereas the increase in amount of galactosyltransferase seems largely to account for the rising yield of lactose during lactation. Studies with pinched-off Golgi membrane vesicles, together with measurements of intracellular chemical concentrations, suggest that beta-glucose and uridine diphosphategalactose do not saturate lactose synthesis and are, therefore potentially regulatory features of this process. Further aspects of lactose synthesis that may offer points of regulation include calcium ions, generation of protons within the Golgi lumen, and the generally rate-limiting nature of the Golgi membrane.

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1. At short incubation times, and under suitable osmotic conditions, the lactose synthesized by Golgi-derived vesicles of rat mammary gland is 85-90% particulate. Evidence is presented for its occlusion within the lumen of the vesicles. 2. Ovalbumin is used as a bulky active-site inhibitor to show that the active site of lactose synthase lies on the inner face of the Golgi membrane. 3. Phlorrhizin and phloretin inhibit lactose synthesis by such vesicles, indicating the presence of a glucose-transport system. 4. The relationship of this topography to the synthesis of N-acetylneuraminyl-lactose and to the secretion of milk sugars is discussed.

1. Tissue concentrations of nucleic acids, protein, fat, water, metabolites and lactose, and the activities of seven enzymes concerned in milk biosynthesis, were measured in the rat mammary gland at closely spaced times before, at and after parturition. 2. Changes are seen in the tissue concentrations of most substances, and several changes are initiated at least during the day preceding parturition. 3. Lactose, which is absent 1 day before parturition, is found in amounts of 12mumoles/g. fresh wt. of tissue at parturition. 4. From the tissue activities before parturition of three enzymes on the biosynthetic pathway of lactose, and, from the small changes observed in their activities at parturition itself, it is concluded that the factors responsible for the appearance of lactose at parturition remain to be demonstrated.