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W-017: The Control of Lactase Activity beyond the Lac-Operon

MIREILLE A. CAPTIEUX¹, TAYLOR E. SWAIN², ALICIA A. MILLS³, LAUREN M. MILLS³, and CARL W. VERMEULEN⁴

1. Univ. of Edinburgh, Edinburgh, Scotland EH16 5AY
2. Jamestown High School, Williamsburg, VA 23185
3. Messiah College, Grantham, PA 17027
4. Science-Projects.com, Williamsburg, VA 23185 USA.

INTRODUCTION

One of the classic pillars of training in molecular biology is the lac-operon. Here it is shown with a few auxilliary controls:

We reasoned, however, that this amounted only to being an "on/off" control of ß-galactosidase (lactase) activity. There must be a level of fine control required of this enzyme to complete the total picture.

We approached this through a series of in vitro and in vivo experiments (panels to the right). We first needed to know if ß-galactosidase (lactase) was indeed susceptible to some form of in vitro inhibition, and, if so, could this be demonstrated in vivo.

In Vitro Methods

Reasoning that either substrate or product inhibition would be the most likely form of control, we added large excesses of various sugars to the lactose analogue ONPG, and then added lactase. If no yellow color developed, that particular sugar was deemed the inhibitor. Lactose was used as a control as it would compete with the ONPG.

Results: Qualitatively, within seconds only lactose and GAL (but not glucose) strongly inhibited the conversion of ONPG to yellow. Our conclusion: the enzyme was controlled by some form of feedback inhibition.

Kinetic Studies

In order to ascertain how the enzyme was inhibited, we employed the Lineweaver-Burk method (LB) which discern allosteric ("A") from competitive ("C") types of inhibition.

Thus, the rates, V, of development of ONP's yellow color were measured, and plotted using double-reciprocal plots of 1/V and 1/[ONPG].

Results: GAL-inhibition was found to be quantitative, and on LB plots straight lines converged at 1/Vmax. This sums to indicate that control was by competitive feedback inhibition.

In Vivo Methods

Since a gal- strain of E.coli lac+ could not digest and thus diminish the amount of galactose present, we chose it to determine the impact of exogenous galactose on lactose digestion. Its results were compared with those of a strain of otherwise identical genetic background. The genotypes are shown at the bottom of this column.

These were grown on lactose-MacConkey agar (MC) both without GAL and with excess GAL (GAL/LAC = 4). Shown here is the excess GAL plate, and the subsequent inspection of their internal concentrations of lactase using SDS-CHCl3 augmented vortex disruption and addition of ONPG with these results:

Results: On the lactose + MacConkey plates, while the gal+ strain produced red colonies on all plates, the gal- strain was only red in the absence of GAL. The non-red gal- cells that had grown in the presence of both LAC and GAL exhibited high levels of lactase activity.

Completed Control Schematic of Lactase Activity

Discussion

This is the newly amended schematic for the overall control of lactase activity. In essence, the genetic level supplies the enzyme, if needed, while the metabolic level fine-tunes the activity of those enzyme molecules to rates commensurate to the cell's need for energy production.

Had galactose only worked as an inhibitor in vitro, the whole process would have been purely academic. However, the significance of this work is that it is operable in living cells.

Of special interest to educators is the fact this builds upon and completes one of the most commonly taught units in high school and college biology. What is more is that all of this is easily applicable to teaching labs in general biology, microbiology, genetics and biochemistry. Of great importance to the high school level is that no toxins are used in this series of lab exercises - no mercurials, no iodoacetates, no cyanide. The inhibitor is simply galactose, which our mammalian bodies readily convert to glucose and catabolize.

Acknowledgments

We wish to thank the E.coli Genetic Stock Center at the Yale University Medical School for their gift of the two E.coli strains used in this work. Most of all we wish to thank our teachers who facilitated this far-flung work: Mr. Scott McKissock of Cambridge Springs High School in Pennsylvania, Mr. Charles Dubay of Jamestown High School in Williamsburg, Virginia, and Mr. Tovia Rosenfeld of Midwood High School at Brooklyn College in New York.

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Students who access this website are encouraged to work collaboratively on original projects with an aim toward making presentations beyond science fair. This is the fourth presentation at the ASM made under this logo. The website is owned and operated by Dr. Vermeulen, emeritus microbiologist from the College of William and Mary in Williamsburg, Virginia.

Post-Acceptance Addendum

Diana Kuruvilla and Lian Liang
Midwood High School at Brooklyn College
Brooklyn, NY

As shown next, in vivo evidence indicates that at GAL/LAC ratios of less than one, the gal- cells behave on Lactose-MacConkey as lactose fermenters. However at ratios above one, they behave as lactose non-fermenters.

As also seen at ratios above one, those same gal- cells hyper-produce lactase while being ineffectual in using it. However, if the MacConkey plates are allowed to stand for several days, all plates of gal- eventually become red indicating a "leakiness" in the gene's activity - something expected of a competitively inhibited system.