The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme.

The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adunyah, J.T. Penniston and E. Carafoli, 1981, J. Biol, Chem. 256, 395 - 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that ot the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+/ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.

A procedure for dissociating Ayre scrape samples.

The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.

Value of screening umbilical cord blood for hemoglobinopathy.

Foremost among the beneficial effects of screening umbilical cord blood is the optimized quality of care that can follow the immediate involvement of an infant with sickle cell disease and his or her family in an appropriate health care system. This is exemplified by the reduction in the case fatality rate of pneumococcal septicemia that has been achieved. Appropriate follow-up of screening also includes transmission of information about the diagnosis of a hemoglobinopathy trait or alpha-thalassemia to affected families and their physicians, with ready availability of education and counseling.

The (Ca + Mg)-ATPase of reticulocytes.

Rabbits were made reticulocytotic by repeated bleeding or by injection of phenylhydrazine. Up to reticulocyte levels of 70%, the baseline activity of magnesium-dependent calcium-stimulated adenosine triphosphatase ( ( Ca + Mg)-ATPase, EC 3.6.1.3.) of the red cell plasma membrane was not significantly changed. Maximal activity in the presence of calmodulin was significantly reduced, the stimulation by the activator falling from about 350% to about 50%.

Evaluation of clinical severity in sickle cell disease.

For a severity classification of sickle cell disease to be accepted, it is necessary that clinicians agree upon relative disease severity between patients. This condition was shown to be satisfied for a randomly selected group of patients evaluated by four persons. All rank correlation coefficients between observer pairs were highly significant. Representative severity indices based on history and recent hospital events also correlated significantly with evaluator ranking. The results show that, in principle, a classification of sickle cell disease patients by severity is possible. Such a classification would be most useful to evaluate the prognostic significance of particular signs or symptoms, or the success of various treatments in affecting severity of disease.

Erythrocyte calcium metabolism. Calcium exchange in normal and sickle-cell-anaemia erythrocytes.

Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.

Calcium exchange and calcium-related effects in normal and sickle cell anemia erythrocytes.

There is an exchangeable calcium pool in both normal and sickle cell erythrocytes, comprising about 10-15% of the total cellular calcium. Sickle cells show increased calcium as compared to normal cells in the oxygenated state. Specific differences between sickle and normal cells which may be associated with this fact are an increased rate of calcium exchange in sickle cells at low external calcium, an increased "leak" of calcium into sickle cells (i.e., phosphate independent exchange), and a pattern of magnesium loss in sickle cells which is consistent with a Mg-Ca exchange diffusion resulting in the increased intracellular calcium in these cells. The exchangeable calcium in sickle cells is more labile, almost all of it being available for re-exchange out of the cell over a short-time-course experiment. Analyses of flexibility and osmotic fragility of sickle cells are consistent with expected effects of increased intracellular calcium.

Water sorption and vapor-phase deuterium exchange studies on methemoglobin CC, SC, SS, AS, and AA.

Five hemoglobins whose genetic relationship to one another involves one set of alleles, hemoglobins CC, SC, SS, AS, and AA, were studied in the Met form. Two different investigations were conducted at 28 degrees C on these methemoglobins within a McBain gravimetric sorption system: sorption of H(2)O vapor and vapor-phase deuterium-hydrogen exchange. For each of the five samples there was close agreement between the per cent hydration of polar sites as determined from sorption studies and the maximum per cent of labile hydrogens that were exchanged during the vapor-phase deuterium exchange study. Both studies measured a slight increase in the number of polar sites accessible to H(2)O or D(2)O vapor for those samples in which the substituent in the sixth position from the N-terminus of the two beta-chains had a positively charged side chain and a slight decrease for those in which the substituent had a negatively charged side chain. The in-exchange of deuterium for hydrogen occurred at a faster observed rate than the out-exchange of hydrogen for deuterium.

Evidence for a carrier-mediated exchange-diffusion entry of calcium into erythrocytes.

The enthalpy of activation of entry of 45Ca into erythrocytes under conditions of zero net transport (19 Kcal/mol), inhibition of entry by N-ethylmaleimide, and transport pH optimum of 7 - 7.5 are consistent with a protein carrier for inward calcium movement. Initial rate analysis shows saturation kinetics and transstimulation, consistent with a carrier-mediated exchange-diffusion mechanism.

Hemoglobin S levels in sickle cell trait individuals.

Changes in Department of Defense regulations now permit persons with sickle cell trait to serve in all service branches. However, for purposes of the regulation, sickle cell trait is defined as 41% or less S hemoglobin. Our screening experience, based on 397 individuals with sickle cell trait, with quantitative scan of cellulose acetate electrophoretic sheets, indicates that 20-40% (depending on definition of terms) of individuals with sickle cell trait would be excluded by this criterion.

Absorption spectra of sperm-whale ferrimyoglobin.

1. Sperm-whale ferrimyoglobin was found to contain 0.308% of Fe, on a dry weight basis, corresponding to a molecular weight of 18130. The solid takes up moisture to an equilibrium state, and was then assayed to contain 0.280% of Fe. 2. Absorption spectra are presented for acidic ferrimyoglobin, Fe(+)(H(2)O), and its conjugate base, Fe-OH, as well as for the fluoride and cyanide complexes, within the range 200-2500mmu. Data for ferromyoglobin-carbon monoxide, Fe(II)-CO, in the visible region are also included. 3. Minor spectral differences have been found between whale and horse myoglobins, particularly in the effect of temperature on the visible-absorption spectrum of Fe-OH.

Glycosylated hemoglobins in heterozygotes and homozygotes for hemoglobin C with or without diabetes.

Glycosylated hemoglobin in red blood cell hemolysates of five patients homozygous for CC, 18 patients with SC condition, and 13 patients heterozygous for Hb C with or without insulin-dependent diabetes mellitus were separated by Bio-Rex 70 chromatography. The various glycosylated components were identified by analysis of the hemoglobin components for ketoamine and phosphate, in vitro glycosylation studies, and by the quantitative differences in the minor components between the participants with and without diabetes. The percentages of Hb A1a + b, Hb A1c, and Hb C1c were significantly increased in the Hb C heterozygote with diabetes. Similarly, the percentages of Hb S1a + b and Hb S1c were elevated in the SC patient with diabetes. It was noteworthy that the levels of these components became normal after adequate control of diabetes. Moreover, the levels of Hb C1c in the CC participants and Hb S1c (Hb S1c/total Hb S) in the SC patients were significantly higher than the Hb S1c levels previously reported in patients with sickle cell anemia. These findings might reflect the fact that CC and SC patients have less severe hemolytic anemia. Moreover, the relative proportions of Hb A1c and Hb C1c were nearly the same in Hb C heterozygotes, which indicated that Hb A and Hb C were glycosylated in vivo to approximately the same extent.