The M-L blood group system and active potassium transport in sheep reticulocytes

Immature and mature red cells from anaemic homozygous and heterozygous low potassium (LK) type sheep were tested for the presence of the L antigen and for active potassium uptake. Evidence was obtained for the presence of L antigen on immature as well as mature cells, but immature cells had a higher intracellular potassium concentration and increased rate of active potassium transport which was only slightly enhanced after sensitization with L antiserum. The red cells which entered the circulation in the later phases of recovery from anaemia were not haemolysed by anti-L as readily as normal cells, but showed normal haemolytic reactions with other blood typing reagents.
It is suggested that, if the L antigen is directly concerned with suppressing part of the potassium pump in mature LK erythrocytes, its effect must be mediated by changes which occur during the final maturation processes of the red cell.     

The relationship between the S system of blood groups and potassium levels in red blood cells of cattle

Red cell potassium concentrations were determined in 5 breeds of cattle. Most cattle had red cells with less than 50 mmol K/litre and although a few Hereford and Friesian cows had levels approaching 70 mmol K/litre, it was only in the Jersey breed that values above this were found. Attempts were made to correlate potassium levels with serological specificity using cattle S system and sheep M system blood typing reagents. Red cells negative for cattle factors S, H', and S were never positive for sheep L; cells positive for H' were usually positive for M, and those positive for S were always positive for L. No obvious relationship was found between potassium levels and serological type, although some consistent trends were noted in Friesians and Herefords. Limited family data indicated that a major difference in potassium levels of red cells in cattle may be controlled by two codomi-nant alleles.     

The effect of sheep anti-L and certain cattle S-system reagents on active potassium transport in sheep and cattle red blood cells

Certain anti-sheep L antisera stimulated active potassium transport in cattle red cells. All cattle red cells tested (red cells from 21 Jersey cows) which had an internal K level of less than 70 mmol/1 were stimulated but those with more than 75 mmol/1 of K (red cells from 7 Jersey cows) were not stimulated. Cattle S-system reagents and isoimmune cattle sera produced by injecting red cells of low-potassium type into cows with cells of high-potassium type failed to stimulate active potassium transport in either cattle or sheep red cells.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

Interactions between earthworm hemolysins and sheep red blood cell membranes

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.     

绵羊红细胞敏感性对补体结合试验的最佳反应时间

正补体结合试验(CFT)是一种经典的抗原、抗体检测方法,其敏感性、特异性均较高,不仅可用于诊断各种传染病,也可用于鉴定病原体,是免疫学上一个应用较广泛的重要试验[1]。在布鲁氏菌病(布病)的血清学诊断方法中,CFT被世界动物卫生组织(OIE)认可为布病血清学确诊的经典标准方法[2]。在CFT实际操作中经常发现,使用不同保存时间的绵羊红细胞(SRBC)滴定同一批次溶血素和补体时,结果存在较大差异,有时效价可能相差     

Na,K-ATPase isoform expression in sheep red blood cell precursors

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.     

On the structure of agglutinated sheep red blood cell membranes

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160–186 Å. It is concluded that two asymmetric membranes are contained inthe elementary period. Lipid phases with preferentialyl oriented hydrocarbon chains are part of the membrane structure. The stacking of membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 Å^?1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts

Summary The exposure of red cell ghosts to external Ca⁺⁺ and K⁺ leads to a rapid net K⁺ efflux. Preincubation of the ghosts for various lengths of time in the absence of K⁺ in the external medium prior to a challenge with maximally effective concentrations of Ca⁺⁺ and K⁺ renders the ghosts unresponsive to that challenge with a half-time of about 7–10 min. Preincubation at a range of K⁺ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K⁺ concentrations of about 500 m or more suffice to maintain the K⁺ channel in a maximally responsive state for at least 60 min. These K⁺ concentrations are considerably lower than the K⁺ concentrations required to make the responsive channel respond with a maximal rate of K⁺ efflux. Thus, external K⁺ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state.The presence of Mg⁺⁺ or ethylenediamine-tetraacetic acid (EDTA) in the K⁺-free preincubation media preserves the responsiveness to a challenge with Ca⁺⁺ plus K⁺. In contrast to external K⁺, the presence of external Ca⁺⁺ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca⁺⁺ while washes with EDTA after exposure to Ca⁺⁺ do not reverse them.In red cell ghosts that contain Ca⁺⁺ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K⁺ is enhanced. The effects of incubation in the presence of Ca⁺⁺ in K⁺-free media are reversed; external Ca⁺⁺ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K⁺-free media prior to a challenge with external K⁺ and internal Ca⁺⁺ does also take place when K⁺-efflux from red cell ghosts is measured by means of⁴²K⁺ into media that have the same K⁺ concentrations as the ghost interior. This confirms that the effects of K⁺-free incubation are due to the modification of the K⁺-selective channel rather than to an inhibition of diffusive Cl^–-efflux.Abbreviation used in text TRIS Tris (hydroxymethyl) aminomethan This paper is dedicated to the memory of Walther Wilbrandt.     

The M-antigen in HK and LK sheep red cell membranes

Summary Red cells of all high-potassium-type (HK) sheep and of more than one half of all low-potassium-type (LK) sheep contained the M-antigen and were hemolyzed by iso-immune anti-M antiserum in presence of a guinea pig serum complement. It was characteristic for the hemolysis of HK red cells by the M-antiserum the all HK cells were ultimately hemolyzed at suboptimal antibody concentrations, provided the time of incubation at 37 °C was sufficiently long. Thus, the M-antigen appears to be expressed on all red cells of an individual HK sheep. The M-antibody was absorbed by HK red cells and their membranes with a high affinity, whereas M-negative LK red cells and their membranes did not bind the antibody. The ratio of the number of antibody units absorbed per cell or membrane to the number of antibody units required for lysis approached unity. The amount of antibody absorbed per membrane was unaffected by ouabain in the presence of ATP, Mg⁺⁺, Na⁺, and K⁺. The M-antigen activity depends on the integrity of the red cell membrane and was not detectable after lyophilization of HK membranes or in the membrane protein solubilized by n-butanol. The major M-antibody activity was found among the high molecular weight plasma proteins and may be attributed to the ₂ M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly -globulin. The relationship between the M-antigen and the Na⁺–K⁺ transport system in sheep red cell membranes is discussed.This work was presented in part at the 53rd annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. 1969.     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.