While macro-and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types -principal and intercalated cells -has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1 -/-mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1 -/-mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.

The invariant active site residue Glu 441 in protein R1 of ribonucleotide reductase from Escherichia coli has been engineered to alanine, aspartic acid, and glutamic acid. Each mutant protein was structurally and enzymatically characterized. Glu 441 contributes to substrate binding, and a carboxylate side chain at position 441 is essential for catalysis. The most intriguing results are the suicidal mechanism-based reaction intermediates observed when R1 E441Q is incubated with protein R2 and natural substrates (CDP and GDP). In a consecutive reaction sequence, we observe at least three clearly discernible steps: (i) a rapid decay (k 1 > 1. Ribonucleotide reductase is an essential enzyme of all living cells and catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides. Several classes of ribonucleotide reductases with different subunit composition and cofactor requirements are known, but they all share a radical-based reaction mechanism (1).The aerobic class Ia ribonucleotide reductase from Escherichia coli is the best characterized enzyme. It consists of two components denoted protein R1 and protein R2, each of which is a homodimer. Protein R1 contains redox-active cysteines essential for catalysis. Cysteines 225, 439, and 462 are located at the active site, where all four physiological substrates (CDP, UDP, GDP, or ADP) can bind. R1 also contains two different allosteric sites that bind nucleoside triphosphate effector molecules. One site regulates the overall enzyme activity, and the other site determines the substrate specificity (2, 3). Protein R2 contains a stable tyrosyl free radical at position 122 and an adjacent dinuclear iron center (4 -6). The tyrosyl radical is essential for catalysis.The separate three-dimensional structures of protein R1 and of protein R2 are known (6 -9). A model-built holoenzyme complex of the R1 and R2 structures indicates that the distance between the active site in R1 and Tyr 122 in R2 is about 30 -40 Å (8). Chains of conserved hydrogen-bonded residues leading from the active site of R1 in the direction of Tyr 122 in R2, and vice versa, have been identified and are believed to be part of a radical transfer pathway between the two sites (1, 6 -9). Mutational analysis of the residues postulated to be involved in radical transfer between R1 and R2 during catalysis supports this hypothesis (4, 10 -14).

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These results demonstrate the existence of a cross-talk between the nitrate-nitrite-NO pathway and the NOS-dependent pathway in control of vascular NO homeostasis.

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATPbinding cassette transporters were also identified as were two subunits of the F 0 membrane part of the ATP synthase.