Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation.

The histidine rich protein II (HRPII) from Plasmodium falciparum has been implicated as a heme polymerase which detoxifies free heme by its polymerization to inactive hemozoin. Histidine-iron center coordination is the dominant mechanism of interaction between the amino acid and heme. The protein also contains aspartate allowing for ionic/coordination interactions between the carboxylate side chain and the heme metal center. The pH profile of heme binding and polymerization shows the possibility of these two types of binding sites being differentiated by pH. Circular dichroism studies of the protein show that pH and heme binding cause a change in conformation above pH 6 implying the involvement of His-His+ transitions. Heme binding at pHs above 6 perturbs HRPII conformation, causing an increase in helicity.     

Alpha,beta-dehydrophenylalanine containing cecropin-melittin hybrid peptides: conformation and activity.

Synthesis and conformational studies of a cecropin-melittin hybrid pentadecapeptide CA(1-7)MEL(2-9), and its three alpha, beta-dehydrophenylalanine (DeltaPhe) containing analogs in water-TFE mixtures are described. DeltaPhe is placed at strategic positions in order to preserve the amphipathicity of the molecule. The wild type CAMEL0 and its three analogs, containing one, two and three DeltaPhe residues namely CAMELDeltaPhe1, CAMELDeltaPhe2 and CAMELDeltaPhe3 respectively were synthesized in solid phase and their conformation determined by CD and NMR. CAMELDeltaPhe2 and CAMELDeltaPhe3 peptides exhibit the presence of 3(10)-helix and beta-turns in the former and only turns in the latter. CAMELDeltaPhe1 peptide was found to have a largely extended conformation. Antibacterial and hemolytic activities of the peptides were also evaluated. CAMELDeltaPhe2 peptide is maximally potent against both Staphylococcus aureus ATCC 259230 and Escherichia coli ATCC 11303. CAMELDeltaPhe1 with a single DeltaPhe at the center shows minimal hemolysis.     

Conformations of peptides containing Z-alpha,beta-dehydroleucine (delta ZLeu). A comparison of Boc-Pro-delta ZLeu-Gly-NHEt and Boc-Pro-delta ZPhe-Gly-NHEt

Two tripeptides of the type Boc-Pro-delta ZX-Gly-NHEt (where X = Leu, Phe) have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. and i.r. spectroscopy. These conformational studies indicated that delta ZLeu, similar to delta ZPhe, has a strong tendency to stabilize folded Type II beta-turn conformations when present at i + 2 position.     

Glutathione as an inhibitor of trypsin induced proteolysis

Glutathione has been shown to inhibit trypsin induced proteolytic activity. A concentration of 6 mM of glutathione was found to completely inhibit proteolysis of 3H-proline labelled underhydroxylated procollagen as a substrate, whereas a concentration of 2.1 mM of glutathione caused 50% inhibition of proteolysis. When azocoll was used as a substrate for trypsin 50% inhibition of proteolysis was achieved with 1.4 mM of glutathione, though a complete proteolytic inhibition was attained at 4 mM glutathione. The results suggest that glutathione may be playing an important role in protein metabolism in a variety of disease and stress states.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Calcium diphosphatidate membrane traversal is inhibited by common phospholipids and cholesterol but not by plasmalogen

Phosphatidate-mediated Ca2+ membrane traversal is inhibited by phospholipids (PL) such a phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin and lysoPC, but not by PC-plasmalogen. Kinetics of Ca2+ traversal through a 'passive' bilayer consisting of OH-blocked cholesterol show competition between PC and phosphatidic acid (PA); it appears likely that a Ca(PA.PC) complex is formed which is not a transmembrane ionophore but will reduce the amount of phosphatidic acid available for the formation of the ionophore, Ca(PA)2. PS and PI may inhibit Ca2+-traversal in the same manner by forming Ca(PA.PL) complexes. We suggest that PC-plasmalogen, with one of the Ca2+-chelating ester CO groups missing, cannot engage in calcium cages, i.e., Ca(PA.PL) complexes, and thus does not interfere with Ca(PA)2 formation. Double-reciprocal plotting of Ca2+ traversal rates in cholesterol-containing liposomes vs. calcium concentration suggests that cholesterol inhibits Ca2+ traversal by competing with Ca2+ for PA. The inhibition does not seem to be caused by a restructuring or dehydration of the membrane 'hydrogen belts' affected by cholesterol; most probably, it is due to hydrogen bonding of the cholesterol-OH group to a CO group of PA; this reduces the amount of PA available for the calcium ferry. The inhibition by sphingomyelin and lysoPC may also be explained by their OH group interacting with PA via hydrogen bonding. The pH dependence of Ca2+ traversal suggests that H[Ca(PA)2]- can serve as Ca2+ cross-membrane ferry but that at physiological pH, [Ca(PA)2]2- is the predominant ionophore. In conclusion, the results indicate that Ca2+ traversal is strongly dependent on the structure of the hydrogen belts, i.e., the membrane strata occupied by hydrogen bond acceptors (CO of phospholipids) and donors (OH of cholesterol, sphingosine), and that lipid hydrogen belt structures may regulate storage and passage of Ca2+.