Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to “drive” cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range −200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.     

A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.     

Painful subacute thyroiditis in Hawaii.

Between 1960 and 1982 we prospectively studied 269 patients with painful subacute thyroiditis to determine the demographic characteristics, seasonality, and natural course of the disease. The mean age for all patients was 37.1 years. The female:male ratio was 6.7:1. At the first visit, disease was bilateral in 69%. No epidemic or seasonal pattern was identified. The mean duration of thyroid tenderness was 2.2 months and that of palpable thyroid lumps was 2.8 months. This time difference, sometimes lasting many months, left a painfree "window" during which the palpable hard residual mass of subacute thyroiditis may be confused with other thyroid problems, especially cancer.     

Highly restricted distributions of hydrophobic and charged amino acids in longitudinal quadrants of alpha-helices

Helix formation in folding proteins is stabilized by binding of recurrent hydrophobic side chains in one longitudinal quadrant against the locally most hydrophobic region of the protein. To test this hypothesis, we fitted sequences of 247 alpha-helices of 55 proteins to the circular (infinite) template (symbol; see text) to maximize the strip-of-helix hydrophobicity index (the mean hydrophobicity of residues in (symbol; see text) positions). These template-predicted configurations closely matched crystallographic structures in 87% of four- or five-turn helices compared. We determined the longitudinal quadrant distributions of amino acids in the template-fitted, sheet projections of alpha-helices with respect to the best longitudinal, hydrophobic strip on each helix and to the N and C termini, interiors, and entire helices. Amino acids Leu, Ile, Val, and Phe were concentrated in one longitudinal quadrant (p less than 0.001). Lys, Arg, Asp, and Glu were not in the quadrant of Leu, Ile, Val, and Phe (p less than 0.001). Significant quadrant distributions for other amino acids and for termini of the helices were also found.     

Author index to volume 167

The N-terminal amino acid sequences of two chloroform/methanol soluble globulins from barley and one form wheat are reported. They are homologous with N-terminal sequences previously reported for α-amylase and trypsin inhibitors from cereals and 2 S storage proteins from castor bean and rape. Three albumins were also purified from Aegilops squarrosa and Triticum monococcum. These had N-terminal amino acid sequences most closely related to the α-amylase and trypsin inhibitors. The relationships of this superfamily of seed proteins are discussed.     

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production

The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. In conclusion: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.     

Class II restriction in mice to the malaria candidate vaccine ring infected erythrocyte surface antigen (RESA) as synthetic peptides or as expressed in recombinant vaccinia

The immune response to three peptides corresponding to the repeat regions of the malaria candidate vaccine ring infected E surface Ag (RESA) were studied. Both antibody responses and lymphocyte stimulation in mice injected with these peptides without carrier were found to be restricted to certain MHC class II haplotypes. Mice bearing IAk were strong responders to all three peptides. Mice bearing IAd were strong responders only to the 3' repeat peptides, the octamer and tetramer. Mice bearing Is or Iq did not respond to any repeat peptides. Remarkably, the pattern of genetic restriction of the antibody response to the entire RESA as expressed in vaccinia indicated that there were no other epitopes besides the three repeats. Because only one class II haplotype (i.e., k) out of five tested responded strongly to this peptide and only two out of five (i.e., k and d) responded to the octamer or tetramer, it may be difficult to achieve a good immune response against RESA in most or all humans.