568 J. T. HOLDEN 
seriously limit the utility of a system as a model for studying transport phenomena. 
Unfortunately, most studies have relied almost exclusively on measurements of 
isotope in cell extracts, occasionally supported by chromatographic evidence showing 
that most or all of the isotope is still present in the original amino acid. Seldom has 
the optical configuration of the amino acid been confirmed or the supernatant fluid 
examined for metabolites of the amino acid. Many of these difficulties can be avoided 
by using a non-metabolizable solute such as a-aminoisobutyric acid!® 76 or by em- 




Atmoles : GLUTAMATE /Smin/20mg CELLS 


EXTERNAL GLUTAMATE (zmoles/ml ) 
Fig. 1. The effect of external glutamic acid concentration on the rate of glutamate accumulation 
by L. avabinosus. Standard uptake conditions as described by HoLDEN AND Hotman®+. The inset 
shows these data plotted according to the method of LINEWEAVER AND BurRK®”. 
ploying a mutant strain incapable of significantly metabolizing the test substance*®. 
The associated movement or counterflow of other cell constituents or metabolites 
during amino acid accumulation, which could have an important bearing on an 
analysis of the reaction mechanism, has seldom been explored in microbial studies. 
While the simple procedures described above generally suffice to demonstrate 
accumulation, decisions regarding the mechanism of accumulation pose vastly 
more difficult methodological problems. For example, direct evidence demonstrating 
the intracellular state of accumulated amino acids so far has proven difficult to obtain 
in most systems. It is anticipated that much more sophisticated techniques will be 
required to detect, measure and identify transport catalysts in cell fractions. 
Effect of external concentration on accumulation rate and capacity 
The effect of external concentration on the L-glutamic acid accumulation rate in 
L. arvabinosus is illustrated in Fig. 1. The data fit a straight line when plotted accord- 
ing to the method of LINEWEAVER-BurRK. Similar results have been observed for 
glutamate accumulation by S. faecalis’, and for the uptake of various amino acids 
by S. cereviseae™ and E. coli!®. Such behavior indicates an interaction of exogenous 
amino acid with a cell component present in limiting quantity. It is clearly recognized 
that this does not permit a choice between the operation of rate-limiting catalysts or 
of internal binding sites. Few investigations include conclusive evidence for the 
participation of catalysts other than those concerned with the supply of energy. 
Only a few examples have been reported in which a limiting rate was not observed 
References p. 592/594 
