Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

The conformational analysis of oligosaccharides by H-NMR and HSEA calculation

The application of 1H-nuclear Overhauser enhancement, 1H-spin-lattice-relaxation-time and 1H-chemical shift measurements for the assessment of the conformational preferences of oligosaccharides are briefly reviewed. It is demonstrated that additivity rules, for the correlation of the chemical shifts of similar hydrogen atoms in different oligosaccharides, can be useful in the conformational analysis of oligosaccharides when the differential chemical shifts are greater than 0.1 ppm. These often can be attributed to specific interunit deshielding of a hydrogen atom by an oxygen atom with which it is in strong nonbonded interaction. HSEA calculations are used to demonstrate that differential chemical shifts of less than 0.1 ppm can have origins that are not significant to the overall conformational preferences of the oligosaccharides which are being compared. Both shielding and deshielding effects can arise from a change in the orientation of a substituent group as the result of the introduction of a sugar on a neighboring unit. It is demonstrated that substituent groups, such as hydroxymethyl and acetamido groups, on occasions, should be treated in HSEA calculations as freely rotating about their linkage to a pyranose ring.     

Complexes between mitochondrial enzymes and either citrate synthase or glutamate dehydrogenase

Experiments performed in polyethylene glycol and with a divalent crosslinker indicate that both mitochondrial malate dehydrogenase and aspartate aminotransferase can form hetero enzyme—enzyme complexes with either glutamate dehydrogenase or citrate synthase. In general, these as previous results indicate that complexes with the aminotransferase are favored over those with malate dehydrogenase and complexes with glutamate dehydrogenase are favored over those with citrate synthase. When the levels of enzymes are low, the only detectable complex is between the aminotransferase and glutamate dehydrogenase. Under these conditions, palmitoyl-CoA is required for complexes between the other three enzyme pairs, however, palmitoyl-CoA also enhances interactions between glutamate dehydrogenase and the aminotransferase. DPNH disrupts complexes with malate dehydrogenase and has little effect on those with the aminotransferase, while oxalacetate disrupts complexes with citrate synthase but has little effect on those with glutamate dehydrogenase. The citrate synthase-aminotransferase complex was favored in the presence of DPNH plus malate, which disrupt the other three enzyme-enzyme complexes. Glutamate dehydrogenase has a higher affinity and capacity than citrate synthase for palmitoyl-CoA. Consequently, lower levels of palmitoyl-CoA are required to enhance interactions with glutamate dehydrogenase. Furthermore, glutamate dehydrogenase can compete with citrate synthase for palmitoyl-CoA and thus can prevent palmitoyl-CoA from enhancing interactions between citrate synthase and either malate dehydrogenase or the aminotransferase.     

Fatty acid synthetase, malic enzyme and other NADP+ binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.     

Liposome-mediated labeling of adrenocorticotropin fragments parallels their biological activity

To test our hypothesis that specific interactions of ACTH peptides with model lipid membranes reflect the biological importance of similar interactions on target cells, we investigated the liposome-mediated labeling of ACTH fragments with the extremely hydrophobic photolabel, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine. Correlations were found between the labeling rates and the agonistic and antagonistic potencies of the peptides for in vitro steroidogenesis and inhibition of a synaptosomal protein kinase. A model for the cross-reactivity between ACTH and opioid peptides is discussed.     

Toxoplasma gondii: decreased resistance to intracellular bacteria in mice

The effect of sublethal inocula of Toxoplasma gondii on the course of listeriosis and salmonellosis in mice was investigated. Intravenous injection of T. gondii 24 hr after inoculation of Listeria monocytogenes increased mortality from 16% (L. monocytogenes alone) to 68% (L. monocytogenes + T. gondii) (P less than 0.001). Multiplication of L. monocytogenes in spleens also was increased significantly in mice given T. gondii. By 3 days after infection, mice that had received T. gondii and L. monocytogenes had approximately 10 times the number of L. monocytogenes per spleen compared to mice receiving L. monocytogenes alone. Similarly, mortality and the number of bacteria in spleens were increased in mice injected with Salmonella typhimurium and then inoculated with T. gondii. An in vitro assay of macrophage listeriacidal activity was used to investigate the mechanism of this decreased resistance. Peritoneal macrophages from mice injected with T. gondii were less bactericidal than macrophages from uninfected mice. Delayed hypersensitivity responses to L. monocytogenes antigen were markedly suppressed in mice injected with T. gondii. T. gondii infection appears to suppress both macrophage and T-lymphocyte function and may result in decreased resistance to infections caused by intracellular bacteria.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.