E. B. BLACK AND J. M. STENGLE 
451 
immunizing rabbits with red cells from various 
species including springbok, blesbok, eland, 
pranghorn antelope, and bison. The antiserum 
against springbok red cells most consistently 
provided anti-Z antibodies, which remained in 
the serum after adsorption with type X erythro- 
cytes. In over 1,000 sheep typed by Ras- 
musen,^^ all were either X, XZ or Z. In 40 
sheep tested, 29 were X, 10 were XZ, and 1 was Z. 
By 1960, the A, C, D, M-L, and B systems had 
been discovered using reagents prepared from 
the sera of sheep, goats, cattle or rabbits immu- 
nized with sheep erythrocytes. The B system is 
very complex and seems to be homologous to the 
bovine B system which will be discussed later. 
The D system with its two phenogroups is the 
only system whose test reagent has agglutinat- 
ing rather than hemolysing properties. The A 
system consists of two phenogroups, A and " — " 
(no A) and the C system has three phenotypes 
C, a, and " (no C).^^ 
The M-L system and its relationship to the 
cation content of the sheep erythrocjrte has be- 
come an important area of research which may 
shed light upon some of the basic mechanisms 
of biologic membrane function. The presence of 
the M antigen is associated with a high red cell 
potassium content (HK) ; whereas, the L anti- 
gen is present in red cells with a low potassium 
content (LK).i^-i=5 In the heterozygous (ML) 
condition in this two allele system, the LK state 
is dominant. HK erythrocytes differ from those 
of the LK type in both active and passive trans- 
port of sodium and potassium. Active transport 
of sodium and potassium is four to seven times 
greater in HK than in LK erythrocytes. Like- 
wise, the Ouabain inhibited sodium and potas- 
sium stimulated ATPase has been shown to be 
more active in HK than in LK cells. When LK 
cells (blood type LL) are incubated in isoim- 
mune anti-Lantisera prepared by immunizing 
HK (blood type MM) sheep with red cells from 
an LK (blood type ML) sheep, the potassium 
influx in the LK cells increases six-fold. In con- 
trast, when HK red cells are incubated in either 
anti-LM or anti-MM antisera, the sodium-potas- 
sium transport characteristics of the cell mem- 
brane are not altered. An experiment by Lauf, 
Rasmusen, Tosteson, et al.^^-i'' showed a 5.5- 
fold increase in potassium pump influx in LK 
cells exposed to anti-Lantiserum. When the 
concentration of anti-L was reduced by succes- 
sive dilutions, a two-fold increase in the potas- 
sium influx was observed at a concentration of 
anti-L antiserum Yoq that of the undiluted anti- 
serum (Figure 2). 
Ouabain is known to inhibit the potassium 
pump. It is thought that one molecule of Oua- 
bain blocks one pump site and that the activity 
of the pump is inversely proportional to the 
number of Ouabain molecules bound to the cell 
membrane. Therefore, the number of potas- 
sium pump sites can be estimated if the number 
of Ouabain molecules bound to the membrane is 
known. In experiments to determine the effect 
of anti-L antisera upon the number of pump 
sites, Ouabain binding was measured in LK 
erythrocytes incubated in anti-L antiserum and 
in non-immune serum (NK-NI). Ouabain bind- 
ing to the erythrocytes incubated an anti-L was 
approximately twice that seen when non-im- 
mune serum was used (Figure 3) . These results 
suggest that anti-L antiserum acts by approxi- 
mately doubling the number of active transport 
sites on the red cell membrane. However, the ki- 
netic properties of the sites must also be altered 
since a 4-7 fold increase in the concentration of 
intracellular potassium is observed. 
Snyder, Rasmusen, and Lauf^^ have charac- 
0.125 0.25 0.5 1.0 
RELATIVE SERUM CONCENTRATION 
Figure 2. — Effect of varying concentrations of anti-L- 
antiserum on the active transport of potassium in 
LK erythrocytes'' 
