574 J. T. HOLDEN 
by both substances. Unlike GALE’s experience, there is little metabolic loss of glu- 
tamate in the absence or presence of these compounds. HORECKER e¢ al.°” and OSBORN 
et al.** have reported that the entry and exit reactions governing galactose accumula- 
tion in E. colz can be distinguished by different sensitivities to DNP inhibition. Some 
of our findings suggest that the exit of amino acid is more readily inhibited than 
entry by DNP*®. 
Studies of temperature dependency generally support the conclusion that amino 
acid accumulation involves processes other than passive diffusion. In most cases a 
drastic reduction of the accumulation rate at o° to 4° (Fig. 2), and temperature 
coefficients in excess of 1.8 in the range between 18° and 35° have been observed?"; 4!. 
Some investigators have studied slow accumulation at low temperatures!*: 71. The 
possible dependence of such uptake on residual energy metabolism has not been 
excluded. Of course, in those cases where accumulation is markedly accelerated by 
providing cells with an oxidizable or fermentable substrate, the finding of anything 
but an elevated temperature coefficient would indeed be a surprise. Thus such 
evidence has limited value in deducing the nature of interactions which the accumu- 
lated amino acid undergoes during its passage into the cell. 
Similar limitations apply in studies of the alteration in amount or rate of accumu- 
lation when the extracellular pH is varied. One would like to know, of course, which 
molecular species reacts most successfully in the uptake process. Unfortunately, the 
pH optimum is a summation of the optima of a number of reactions including those 
of the energy-producing reactions and, therefore, provides little information about 
the properties of the catalysts which react directly with the amino acid. In a number 
of cases?7; 4 the optimum pH for maximum accumulation corresponds to the pH at 
which the fermentation rate is most rapid. On the other hand, lysine accumulation 
in S. faecalis?’ increased steadily as the pH was raised to 9.5, which is very close to 
the isoelectric point of this amino acid. Tryptophane uptake (isoelectric point 5.9?) 
in E. coli showed a maximum at pH 8.0 to 8.57. 
Retention and exchange of accumulated anuno acids 
One of the striking observations reported initially by GALE was that little or no 
accumulated glutamate was lost from S. faecalis during incubation in water or buffer 
at 37°. A slow leakage was observed with S. aureus which could be reduced markedly 
by providing the cells with glucose*!. As shown in Fig. 3 glutamate accumulated by 
L. avabinosus similarly is retained with little loss for long periods in water or buffer. It 
is readily eluted (by exchange) from the cell when extracellular glutamate is present 
but only at an elevated incubation temperature. This shows that a source of energy 
is not required to maintain a pool even when gradients of several thousand- fold 
exist, and, for the case of suspension in water, that severe osmotic stress does not 
significantly diminish pool retention. In contrast, Gram-negative bacteria rapidly 
lose accumulated amino acids when suspended in water! !? probably because of an 
inferior ability to oppose unfavorable osmotic forces. This is suggested by the finding 
that small pools of accumulated amino acid are lost very slowly from E. coli incubated 
in buffered media at 25° and that progressively larger fractions of the pool are eluted 
as the medium is diluted with water!. In C. utilis Cowiz AND MCCLURE” observed 
that endogenously synthesized amino acids are not removed from the cell by water 
References p. 592/594 
