Search

Enhancement of immune lysis of HK (MM) and LK (LM) sheep red cells by a high K+ medium did not require that the K+ be present during the action of rabbit hemolysin and complement on the cells. Preliminary exposure of these red cells to a high K+ buffer sufficed, and the K+ effect withstood vigorous washing of the treated cells. Enhancement of immune lysis by a high K+ medium was time, temperature and concentration dependent. It was also 80 to 90% inhibited by 10-4 M ouabain and much less effectively by equimolar G-strophanthidin, the aglycone of ouabain. The transition step from E* to ghost was not demonstrably affected by a high K+ medium. The ability of high concentrations of Rb+ or Li+ to enhance immune lysis was confirmed. Washing experiments suggested that these cations have lesser affinity for relevant sites on the red cell than K+, in the order K+ τ; Rb+ τ; Li+. The Rb+ and Li+ effects on lysis were also antagonized by ouabain. Although these phenomena suggest certain similarities to the stimulation of the traditional red cell Na+-K+ pump, several important differences exist. It is suggested that the K+-induced increment in immune lysis is not directly related to pump activity but possibly to conformational changes in the cell membrane influencing the effectiveness of complement.

The antigens reacting with cold-agglutinin antibodies and present in the red cell membranes of human red cells were found in the water-phase when the washed membranes were extracted with n-butanol. The presence of these antigens was demonstrated by agglutination inhibition, complement fixation, and antibody inhibition as determined by the C1 fixation and transfer test. Although antigen and antibody were not able to react at 37°C when the antigen was present in the intact cell, after solubilization, antigen and antibody reacted equally well at 37°C and at 0°C. The amount of anti-I inhibiting activity present in extracts from adult and cord cells was roughly the same, although the amount of antibody fixed to the intact cord cells was less with cord cells.

1. 1. The effect of isoimmune (anti-L) serum on the Na+-activated ATPase activity of low-K+ (LK) type red cell membranes was studied at low ATP concentration. 2. 2. The L-antigen-antibody reaction results in a marked increase in the velocity of Na+-dependent ATP hydrolysis. 3. 3. The response of anti-L-stimulated Na+-ATPase in homozygous LL (low-K+) membranes to K+ resembles that of low-K+ rather than high-K+ (HK) membranes.

L-antigen-positive low potassium type (LK) sheep red blood cells (SRBC) metabolically depleted by (A) starvation in glucose-free media, (B) incubation in 2-deoxy-D-glucose, or (C) exposure to iodoacetamide (IAA) were more susceptible to immune-hemolysis by ovine anti-L serum and rabbit serum complement (RSC) than control LK SRBC. The magnitude of this effect depended on the concentrations of L-antiserum and RSC used. Although all three treatments leading to enhanced immune-hemolysis of LL or LM (LK) SRBC also resulted in a loss of intracellular adenosine triphosphate [ATPli, the data do not permit conclusion of a direct correlation between immune-hemolysis and [ATP]i in these cells. Restoration of [ATP]i in glucose-containing media of LK SRBC previously metabolically depleted by treatments (A) or (B) was complete within two hours; it was followed, however, by a much slower and only partial return of immune-hemolysis to control values. The depletion induced changes appear to promote binding of RSC to, and/...

Erythrocytes of 19 sheep comprising 3 genetic types were studied for susceptibility to lysis by constant amounts of rabbit hemolysin and human complement. The sheep were classified as low K+ (LK) or high K+ (HK) by red cell K+ concentrations and then as antigenic types LL, LM or MM by detection of L and M erythrocyte antigens with specific antisera. The three genetic types of red cells consistently yielded differing levels of hemolysis in the following order of sensitivity: LK (LL) τ; LK (LM) τ; HK (MM). These differences could not be overcome by higher doses of antibody, were observed with both IgM and IgG hemolysin, and were not attributable to differing rates of hemolysis. A high K+ medium enhanced immune lysis of HK (MM) and LK (LM) cells but not LK (LL) cells. When the relationship of immune lytic potential of red cells to their sphericity and rigidity was examined by microscopic morphology, osmotic fragility and cellular deformability, heterogeneity was encountered within genetic types. The practical importance of these genetic differences in sheep erythrocytes for laboratories performing complement assays as a clinical service is demonstrated by comparative CH50 titers on representative human sera.

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.

Publisher Summary This chapter discusses the action of isoimmune anti-M and anti-L on active potassium transport in high potassium (HK) and (LK) sheep red cells. Active and passive transport of sodium (Na) and potassium (K) are different in HK and LK sheep. Active transport of these ions and the Na + - K-stimulated and ouabain sensitive ATPase activity are about four times greater in HK red cells as compared to LK red cells. These cell types are also different with respect to their antigenic properties. Red cells of all HK sheep possess the M antigen whereas red cells of some LK sheep do not possess the M antigen. LK cells contain the L-antigen, which is absent in HK red cells. Anti-M serum has no effect on K-transport in HK and LK sheep red cells. Anti-M lyses HK red cells in the presence of guinea pig complement. HK red cells and their membrane bound this antibody with a high affinity while homozygous LK cells do not absorb anti-M.

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 β2 M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly γ-globulin. The relationship between the M-antigen and the Na(+)-K(+) transport system in sheep red cell membranes is discussed.

This paper reports on several biochemical characteristics of the M antigen in membranes of high-potassium sheep red cells. 1. 1. The M antigen was solubilized by sodium deoxycholate and retained its activity following removal of detergent. 2. 2. Membrame proteins and lipids were separated by gel filtration in deoxycholate and the M antigen was shown to reside only in the protein fraction, but at reduced levels. 3. 3. Antigenic activity was enhanced by recombination with lipid, and both phospholipid and cholesterol were required for full activation. 4. 4. Treatment of whole M-positive membranes with 2-mercaptoethanol and iodoacetamide resulted in a slight decreased of the M-antigenic activity but completely inactivated the M antigen when performed in the presence of 6 M guanidine hydrochloride. The results indicate that the antigen is bound to the membrane primarily through hydrophobic forces. We suggest that portions of the antigen are relatively buried in the membrane matrix, and are inaccessible to small molecular weight reagents.

This paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K(+)-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K(+)-pump stimulating antibody present in the anti-L serum.