82 NITROGEN METABOLISM 



obtained by centrifuging samples of the whole cell suspen- 

 sion in graduated tubes and the volume of the cellular con- 

 stituents was calculated by assuming that they were mainly 

 proteins whose specific volume is 0-70. Subtraction of the 

 volume of the cellular constituents from the volume of an 

 equivalent amount of whole cells yields a value for the 

 'internal free-space', i.e. the internal environment [5]. 



The amino-acid decarboxylases are enzymes of high 

 specificity (p. 27), consequently this procedure will esti- 

 mate only amino-acids which are initially free in the cells or 

 which are liberated from compounds that are easily decom- 

 posed during the assay procedure, e.g. by heat or by other 

 enzymes present in the decarboxylase preparations. Whilst 

 enzymic decomposition during the assay procedure has not 

 been ruled out, it is unlikely that they arise by the decompo- 

 sition of heat-labile compounds since the same amount of 

 amino-acid is found in cells disrupted by heat as in those 

 made permeable by treatment with tyrocidin or phenol [13]. 

 In the absence of evidence to the contrary the internal 

 amino-acids are regarded as being 'free' in the sense that 

 they are chemically uncombined. 



Internal amino-acids in Gram-positive organisms 



Washed suspensions of twenty-seven organisms com- 

 prising thirteen genera were prepared from cultures which 

 had been grown on a medium rich in amino-acids. The 

 experiments were restricted to the six amino-acids for which 

 specific decarboxylases were then known. Free amino-acids 

 w^ere found only in Gram-positive organisms, yeasts as well 

 as bacteria, and none were detected in Gram-negative 

 bacteria. The yeasts contained high concentrations of all 

 six of the amino-acids whereas the bacteria contained only 

 lysine and glutamic acid in appreciable amounts [18]. 



Absorption of amino-acids by washed cells 



The factors controlling the absorption of lysine and 

 glutamic acid have been studied in experiments with washed 

 cell suspensions of Strep, faeca lis and Staph, aureus {Micro- 

 coccus pyogenes var. aureus), both of which are exacting 



