80 



Cellular Structure and Activity 



ciated with glycolysis appear, on the other 

 hand, to be more soluble* than those asso- 

 ciated with decarboxylation and oxidation 

 (cf. Dixon, '49). 



In addition to enzymes, many small-mole- 

 cule substances such as ATP and acetyl- 

 choline have been considered to be at least 

 in part bound in the normal living cell (cf. 

 Caspersson and Thorell, '42). 



In a general fashion, it may be fruitful 

 for the cell physiologist to regard those en- 

 zyme systems which are fixed to insoluble 

 units of cell homogenates as being of pri- 

 mary importance in the regulatory activities 

 of cells. A notable exception to this general- 

 ization would be the apparent local ac- 

 tivity of phosphorylating mechanisms at the 

 cell surface (Rothstein and Meier, '48). 



Thus a discussion of the control of cellular 

 metabolism should not be limited to test tube 

 factors, but must also include factors relating 

 to the discrete structural organization of en- 

 zyme systems. Basically it might be said 

 that variations in metabolic intensity are 

 due to differences either in molar enzyme 

 concentration or in enzyme activity. But 

 molar enzyme concentrations are rarely 

 known and, under the heading of enzyme ac- 

 tivity, it will obviously be necessary to con- 

 sider spatial orientations as well as material 

 supply and availability of cofactors. 



DIFFERENCES IN ENZYME 

 CONCENTRATION 



The rate at which an enzymatic reaction 

 proceeds may be limited either by the chem- 

 ical reactants in the usual sense, or by the 

 catalytic system. This fact was first clearly 

 established by Michaelis and Menten ('13) 

 in studies on the inversion of cane sugar. Re- 

 cent work has shown that the rate of oxygen 

 uptake may be regulated by the concentra- 



* The word solution is poorly defined in the bio- 

 chemical literature. Due to the lack of precise usage 

 of words by enzymologists, it is difficult to decide 

 whether an "extract," for example, means a solu- 

 tion of the substance in question or whether it is a 

 suspension of granules. Enzymes are here desig- 

 nated as soluble if they have been prepared and 

 crystallized by relatively mild methods. It should 

 be understood that the so-called soluble enzyme 

 may be loosely associated with structures insoluble 

 in the cell or, on the other hand, enzymes difficult 

 to separate from insoluble material may, under cer- 

 tain conditions, be floating freely. Greenstein et al. 

 ('49) record an interesting example of a change of 

 type of enzyme, more liver glutaminase appearing 

 in insoluble fraction with the onset of cancerization 

 while the activity of the soluble enzyme diminishes. 



tion of electron donor (cytochrome c), the 

 concentration of electron acceptor (oxygen), 

 or the concentration of enzyme (cytochrome 

 oxidase). f In this section we shall consider 

 the possibility of variations in activity result- 

 ing from variations in the molar concentra- 

 tion of a given enzyme — necessarily assuming 

 that substrates and cofactors are not limiting 

 in the cell or at localized regions in the cell. 

 Unfortunately, this assumption is not sup- 

 ported by clear-cut evidence at the present 

 time. 



Assays of enzyme content of cells are car- 

 ried out routinely by homogenizing the tis- 

 sue to break down the cell structure, adding 

 appropriate amounts of substrate and co- 

 factors, and then measuring the rate of the 

 reaction in question. Under these conditions 

 one expects that total enzyme content will 

 be determined, any control by cell structure 

 having presumably been abolished by the 

 homogenization procedure. 



Large numbers of studies made with this 

 technique have shown that adult tissues pos- 

 sess distinctive enzyme patterns, differing 

 markedly in ' amounts of various enzymes 

 per unit of tissue. In many cases the differ- 

 ences are plainly correlated with the func- 

 tion of the tissue — for example, high cholin- 

 esterase activity in nerve tissue, high apy- 

 rase activity in muscle, high phosphatase 

 activity in kidney. Under pathological con- 

 ditions enzyme patterns are significantly al- 

 tered (cf. Greenstein, '47). 



The activity of enzymes as determined in 

 test tube assays is obviously less meaningful 

 than activity as deduced from metabolic 

 studies on whole cells. Technical difficulties 

 have prevented such comparisons from be- 

 ing frequently made, but where they have 

 been made (particularly in the field of 

 oxygen uptake), wide discrepancies have been 

 revealed. The work of Spiegelman and Stein- 

 bach ('45), showing a large increase in cyto- 

 chrome oxidase activity on homogenization 

 in frogs' eggs, is an example of this. As a 

 matter of fact, it seems reasonable to assume 

 that most cells of adult organisms ordinarily 



f Activity of an enzyme is usually expressed as 

 rate of substrate change per unit; but the appropri- 

 ate unit basis on which to calculate activity is a 

 difficult matter to decide. Some authors use a 

 standard number of cells, some dry weight, some 

 nitrogen content. Since enzymes are probably local- 

 ized within cells, it is probable that any base 

 unit is valid only in the most general sense, the 

 most specifically valid being the cellular unit, if 

 it can be clearly defined (cf. Davidson and Leslie, 

 '50). 



