Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.     

Phylogeny of mitochondrial DNA clones in tassel-eared squirrels Sciurus aberti

The tassel-eared squirrel, Sciurus aberti , includes six subspecies which occupy restrictive and apparently identical habitats in Ponderosa pine forests in the south-western United States and Mexico; the strict habitat requirement of this species is based on dietary requirements which are only fulfilled in these forests. To examine evolutionary relationships among certain subspecies of S. aberti , we obtained estimates of nucleotide diversity within subspecies as well as nucleotide divergence between subspecies using mitochondrial DNA (mtDNA) analysis. Restriction site polymorphisms were identified in samples of the four US subspecies: S. a. aberti (Abert), S. a. kaibabensis (Kaibab), S. a. ferreus (Ferreus), and S. a. chuscensis (Chuska) Fourteen mtDNA clones were resolved that were, with one exception, uniquely subspecific. Dendrograms constructed by neighbour-joining and maximum parsimony methods revealed two major assemblages: (1) an Abert/Kaibab group; and (2) a Ferreus/Chuska group. The Abert vs. Ferreus clones exhibited the greatest net nucleotide divergence, with a lineage separation estimate approximating 572 000 years ago assuming a nucleotide substitution rate of 7.15 × 10^-9/year/site. Five out of ten Chuska squirrels shared a clone with one Abert sample; the relative sizes of these two populations and their respective ranges as well as their close proximity support the proposal for relatively recent intermixing of Abert and Chuska populations resulting in what appears to be Abert → Chuska migration. Nucleotide diversity within subspecies ranked as Kaibab < Ferreus < Abert < Chuska; the relatively high diversity for the Chuska sample is based on the apparent introgression of Abert mtDNA. The relative diversity exhibited by Kaibab, Ferreus and Aberti samples corresponds to the range size of the respective subspecies.     

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

Five-stranded β-sheet sandwiched with two α-helices: A structural link between restriction endonucleases EcoRI and EcoRV

Examination of crystal structures of restriction endonucleases EcoRI and EcoRV complexes with their cognate DNA revealed a common structural element, which forms the core of both proteins. This element consists of a five-stranded β-sheet and two α-helices packed against it and could be described as α–β sandwich in which helices and β-strands lie in two stacked layers. While the spatial structure of this α–β sandwich is conserved in both enzymes, there are no detectable similarities between amino acid sequences except of a few residues involved in active site formation. Probably, other restriction endonucleases which have similar organization of the active site might possess similar structural element regardless of DNA sequence recognized and recognition elements in the enzyme used. © 1994 Wiley-Liss, Inc.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonns reinhardtii

Summary The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 by staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19–24 by in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.     

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-inactivating proteins (RIPS), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y1 14F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A₁ form of Slt-IA (wild-type-). The same general relationships held: relative to wild type-, Y114F- was attenuated about 7-fold, and Y114S- about 300-fold. Tryptic digestion profiles of the mutant proteins were similar to those of the corresponding wild-type, indicating that the amino acid substitutions had not caused major alterations in conformation. We conclude that Y114 plays a significant role in the activity of Slt-IA, one which is quantitatively similar to that of Y77, and one which is predicated on the presence of both its weakly acidic phenolic hydroxyl and its aromatic ring.     

Gastrin and cholecystokinin in the Eastern Grey kangaroo, Macropus giganteus giganteus

Gastrin and CCK are believed to have a common ancestor. The gastrin structure has probably evolved from CCK-like peptides at a stage later than the amphibians. To trace the evolution of gastrin and CCK we have determined their structures in an Australian marsupial, the Eastern Grey kangaroo. The brain CCK was identical to CCK-8 of most mammals. The larger form of kangaroo gastrin was a 33mer with the sequence pELHPQDLPHLMTDLSKKKGPWQEEDAAY(SO₃)GWMDF-NH₂. The 11 italic residues indicate differences from human gastrin. Gastrin-15 and gastrin-16 comprised about 70% of the total immunoreactivity and resulted from cleavage after the second and third residue, respectively, of the unusual tribasic cleavage site. The smaller forms of kangaroo gastrin differ from most other mammalian gastrins in that the N-termini are not blocked with a pyroGlu moiety. Unlike other gastrins, kangaroo gastrin is more than 95% sulfated. The present study indicates that the gastrin structure, as defined by having a Tyr at position 6 from the C-terminus, evolved before the marsupials diverged from the other mammals 130 million years ago.