Plant Physiol. (1971) 48, 366-370 An Examination of Methods Used to Assay Potato Tuber Invertase and Its Naturally Occurring Inhibitor' Received for publication January 18, 1971 E. E. EWING AND MARTHA H. McADO2 Department of Vegetable Crops, Cornell University, Ithaca, New York 14850 ABSTRACT inhibitor complex is actually of low dissociability, then the equations used workers Confirming an earlier report, it was shown that the en- by previous (5, 9, 16) would be in inhibitor of serious error (2, 4). Therefore, it seems worthwhile to examine dogenous potato tuber invertase forms an es. further the dissociability of the enzyme-inhibitor sentially undissociable complex with the enzyme. Conse. complex. quently, several previous analyses of potato tuber invertase MATERIALS AND which were based on equations derived for highly dissociable METHODS enzvme-inhibitor complexes are presumed to be in serious Crude extracts from potato (Solanum tuberosum L.) tubers error. The complex formation proceeded slowly, requiring were prepared for assay as described by Pressey and Shaw approximately 1 day to reach completion at 2 C, and 1 hr (14), including the use of an Acme Juicerator for homogeni- at 37 C. Allowing complex formation to reach completion zation. A shortened form of the procedure outlined by Pressey before assaying enzyme activity did not affect the noncompeti. (12) was followed for partial purification of invertase inhibitor, tive nature of the inhibition. using only the acid precipitation, fractionation with ammonium Contrary to previous reports that inhibitor could be se- sulfate, adsorption on alumina gel, and Sephadex G-100 steps. lectively inactivated through foaming in a blender, both en- Invertase purification was accomplished by the procedure of zyme and inhibitor appeared to be denatured by such treat- Pressey (12), except that crude extracts were subjected to ment. Foaming accomplished by passing nitrogen gas bubbles longer foaming periods in an attempt to inactivate the inhibitor through extracts gave more favorable results. more completely. Enzyme and inhibitor preparations were buffered with acetate at pH 4.75. Pressey's (11) definition of invertase activity was adopted whereby 1 unit is that amount of enzyme which liberates 1 ,umole of reducing sugar per hr under the conditions of the assay. Pressey's (11) assay was employed with slight modifications. The purposes of this communication are to point out con- The volume of the incubation mixture was reduced to 1 ml. flicts present in published reports of a naturally occurring The mixture was 0.1 M with respect to acetate buffer, pH 4.75, inhibitor of potato invertase; to support the view that the in- and 0.25 M with respect to sucrose. This sucrose concentration vertase-inhibitor complex is of low dissociability; to demon- gave the optimal reaction rate for the range of enzyme con- strate that the complex has an extremely slow rate of formation, centrations used in our experiments. The assay was performed especially at low temperatures; and to examine the degree to at 37 C for 1 hr. The reaction was linear with time throughout which the inhibitor is selectively inactivated by foaming. the assay period. One milliliter of copper reagent (17), which Schwimmer et al. (16) concluded that there was evidence for completely stops invertase activity, was added to terminate the the presence of an endogenous inhibitor of invertase activity reaction. Tubes were immediately heated for 20 min in a in potato tubers. Pressey (11, 12) convincingly demonstrated boiling water bath. After cooling, 1 ml of arsenomolybdate that such an inhibitor was present, reported on its partial color reagent (10) and 7 ml of water were added. Solutions purification, and characterized the inhibitor as a protein with were then mixed and centrifuged 10 min at 500g, and their a molecular weight of about 17,000. By means of Lineweaver- absorbance was read at 520 nm. Boiled enzyme blanks in- Burk plots, he showed that the inhibition was noncompetitive. cluded as controls did not vary significantly in absorbance Several workers (5, 9, 16) applied equations based on non- from reagent blanks. competitive, highly dissociable enzyme-inhibitor complex for- Foaming of solutions was performed with a VirTis blender mation to determine the amount of invertase present in tuber (12, 14) set on medium speed. The homogenizing flask was kept extracts. Pressey (11) mentioned that the reciprocal of enzyme in a water bath at 37 C during blending. An alternative method activity plotted against inhibitor concentration after the of foaming employed N2 gas bubbled into 0.5 ml of extract method of Dixon (1) gave a straight line. This would indicate through a hypodermic syringe needle, the end of which was a highly dissociable complex. However, a later paper (12) stated, cut off square. (Diameter of the needle did not appear to affect without presenting data, that the enzyme-inhibitor complex results.) All solutions foamed were 0.1 M in acetate buffer, pH showed characteristics of low dissociability (6). If the enzyme- 4.75. To compensate for evaporation during foaming, solutions were restored to their original volume by adding a suitable volume of the acetate buffer before assay. ' Supported by Hatch funds granted to Cornell University. Paper 611 of the Department of Vegetable Crops, Cornell Uni- versity. RESULTS 2Present address: 505 W. University Parkway, Baltimore, Md. From previous work (6, 7) with naturally occurring, protein- 21210. like inhibitors, it seemed possible that the combination of in- 366 Plant Physiol. Vol. 48, 1971 POTATO TUBER INVERTASE AND INHIBITOR 367 vertase with the endogenous inhibitor would proceed at a rate sufficiently slow to be measured. To test this possibility, 80 partially purified inhibitor was added to partially purified en- 6 zyme at various intervals prior to the actual invertase assay and preincubated at 2 or 37 C (Fig. 1). To check enzyme I60 stability, the enzyme was also preincubated in the absence of 4 inhibitor. With no preincubation, the amount of inhibitor used 0- A40 -2 reduced enzyme activity by 18%. Preincubation at 2 C further . reduced the activity to only 49% of the control, but consider- 2 I able preincubation time was required to reach the maximal A20 inhibition. For example, 6 hr of preincubation gave inhibition -a about half-way between that obtained without preincubation 1!... L. and the maximal level. At 37 C maximal inhibition was ob- 0 5 10 15 20 25 tained with much shorter preincubation time. Thus 1 day at Inhibitor, pi per 0.846 units invertase 2 C and 1 hr at 37 C each gave 49% of the control activity. FIG. 3. Effect of various amounts of inhibitor added to a given Presumably much of the inhibition which was noted in the quantity of enzyme. Results expressed as reciprocal of reaction absence of a preincubation period or following short preincu- rate ( ) and as percentage of inhibition (---). Vertical line at bation reflects formation of the enzyme-inhibitor complex each point denotes standard error (20 replications). which occurred during the 1-hr assay at 37 C. In spite of the longer preincubation time required at 2 than at 37 C, the lower temperature seemed preferable because the enzyme showed a 80 ~ i = 25 pi gradual loss of activity when preincubated alone at 37 C (Fig. 1). Based on these results, in subsequent experiments mixtures 60 of enzyme and inhibitor were routinely preincubated for 2 days at 2 C before assaying. The preincubation period was extended 0- to 3 or 4 days when working with extremely dilute solutions of .2_ 40 enzyme and inhibitor. The instability of enzyme at 37 C raised the question of q2nLu I 1.00' Enzyme only 0 0.4 0.8 0.75 Invertase, units/sample 0.50 - Enzyme + inhibitor FIG. 4. Percentage of inhibition by a given quantity of in- 0 1 2 3 hibitor for varying amounts of invertase. Vertical line at each .CD E Pre-incubation time at 2', days point denotes standard error (four replications). Enzyme only inhibitor stability under these conditions. Therefore, inhibitor solution was placed in a shaker bath at 37 C for various inter- -____!__ ___ ____ _ _ _ _ _ __ ______ _ ____4 vals up to 4 hr. Enzyme was then added, and the mixture was + inhibitor preincubated and assayed. It was found that even 4 hr at 37 C 3 4 had no detectable effect on inhibitor activity (data not shown). Pre-incubation time at 371, hrs Since Pressey (11) made no mention of a preincubation of FIG. 1. Effect of preincubation time and temperature on in- enzyme and inhibitor, it seemed desirable to repeat the experi- vertase reaction rate in presence and absence of inhibitor. Par- ments from which he concluded that inhibition was non- tially purified enzyme and inhibitor preincubated at 2 C (top) or competitive. The resulting Lineweaver-Burk (8) plots (Fig. 2) 37 C (bottom) for times indicated before assay. Each point repre- verified Pressey's (11) findings of noncompetitive inhibition. sents the mean of six replications. Standard error, 0.01 ,umole/hr. The value of 5.7 mm sucrose for the Km was in good agree- Note the difference in time units used for 2 and 37 C. ment with his values of 6.1 mm (11) and 5.3 mm (12). The next question examined was the dissociability of the enzyme-inhibitor complex. To investigate this, the concen- tration of enzyme was held constant, and the amount of added inhibitor was varied. The results were plotted in two ways: (a) with inhibitor concentration plotted against the reciprocal of the reaction rate, and (b) with inhibitor concentration plotted against percentage inhibition. If the complex is highly dissoci- able, the first method of plotting should produce a straight line and the second method should result in a pronounced curve.