6o6 V. T. NACHMIAS AND J. M. MARSHALL, JR. 



reaction to the cell surface, and was not, as had been assumed previously, 

 a simple matter of the cell engulfing droplets of the medium. Schumaker's 

 kinetic studies showed also that the binding reaction was not affected by 

 cooling or by metabolic inhibitors, although the later stages of vesicle 

 formation were readily blocked. Additional evidence from several sources 

 suggested that the mechanism of the binding reaction w'as electrostatic [4, 

 5, 6] and that the receptor substance was the mucous coat which covers 

 the amoeba. In the work here reported, the mechanism of the reaction was 

 studied by comparing the pH dependence of binding for two closely 

 related proteins, ferritin and methylated ferritin. Binding studies were 

 done on living amoebae {Chaos chaos or Pelomyxa carolmensis) and the 

 results were confirmed by electron microscopy. The same proteins were 

 used in further studies on the changes which occur within the cell after 

 uptake. 



Methods 



At the outset, it was found that protein binding by the cell surface 

 could be "uncoupled" from the remaining stages of pinocytosis by 

 working at 5° [2]. Starving amoebae were rinsed in cold water. By this 

 treatment, the cells were rounded up, cytoplasmic motion was suppressed, 

 and contaminating ions were removed. The amoebae were then pipetted into 

 the cold protein solution, left for 3 to 5 min., and washed in the cold to 

 remove all unbound protein. The washing procedure made it possible to 

 test the reversibility of the surface binding at different pH values [5]. Some 

 cells were fixed at this point in buffered osmium tetroxide, and were 

 embedded and sectioned in epoxy resin for electron microscopy. Others 

 were allowed to warm to room temperature ; in such cells, the complete 

 pinocytosis sequence occurred despite the delay, and the protein carried 

 into the pinocytosis vesicles was only that previously bound to the cell 

 surface and not removed by washing. As a result, it was possible to follow 

 the changes which occurred within the vesicles more clearly than when 

 " bulk" pinocytosis was induced at room temperature. Cells treated in this 

 way were kept in their normal medium for i to 48 hr. after uptake, and were 

 then fixed, embedded, and sectioned. 



PROPERTIES OF FERRITIN AND METHYLATED FERRITIN 



Ferritin was isolated from horse spleen by ammonium sulphate 

 precipitation followed by crystallization in the presence of cadmium 

 sulphate [7, 8]. It has some unusual properties [9, 10], which made it 

 especially suitable for this study. The unit particle of ferritin, which is 

 94 A in diameter, consists of a protein coat surrounding an ordered cluster 



