1. Alkaline Phosphatase Activity and Location in Lumbricus terristris
Sam Steckbauer, Taylor Gut, Sadie Peters, Austin Baetke
Introduction
Alkaline phosphatase, commonly known as ALP, is an enzyme that is found in all living
organisms with the purpose of dephosphorylating molecules in various tissues. The activity of
alkaline phosphatase is optimum in an alkaline environment, as its name suggests. Overall,
ALP is a heat stable enzyme but varies by tissue and organism. ALP is found throughout the
body but higher concentrations are found in select tissues. These tissues include intestinal
epithelia and the placenta. Lower concentrations are found in the kidney and developing bone
and even lower concentrations are found in the liver, lungs, and spleen.
Several experiments were conducted to determine if earthworms (L. terristris) could
serve as a model organism to further the research of Slosaflu. The goals of the experiments
were to determine if the distribution of ALP in tissues of earthworms and mammals were similar,
whether detected phosphatase activity is actually due to ALP, and if the biochemical properties
of ALP in earthworms and mammals are similar.
Results
Histochemical stains were performed to determine location of ALP in L.
terristris. Pharynx, esophagus, intestine and crop/gizzard tissues were soaked in Napthol AS-
MX phosphate and fast blue RR salt which turned the tissue blue where ALP was
present. Pharynx and esophagus cross sections of L. terristris detected little to no ALP present
after being stained. Conversely, intestine and crop/gizzard cross sections showed an abundant
amount of ALP as seen in blue in Figure 1a in the gizzard epithelium layer. In Figure 1b ALP
was found in the epithelium lining the coelom and Typhlosole of the intestine.
In order to quantify location of ALP activity, a p-nitrohpenylphosphotase (pNPP) enzyme
assay was conducted. The end solution would appear yellow at approximately pH 10 if ALP

2. was present. All four L. terristris tissues were individually centrifuged with pNPP and a buffer of
pH 10.5. After 15 minutes in a water bath at roughly human body temperature (37˚C), the
absorbances were read. These absorbances were compared to a purified p-nitrophenol
standard curve to determine simple enzyme activity. It was found that crop/gizzard followed by
intestines showed the highest absorbance and therefore highest ALP activity as shown in Figure
2. Esophagus and Pharynx also showed ALP activity but at much lower levels.
After determining simple activity and location of ALP, multiple enzyme assays were
conducted to determine biochemical properties of L. terristris ALP. Bradford assays were
performed alongside these biochemical assays in order to account for differences in protein
concentration. Due to the location of enzyme activity at pH 10.5, it was deemed crucial to first
explore specific enzyme activity at varying pH in the crop/gizzard and intestines. Crop/gizzard
was added to pNPP at pH 8.5, 9.5, 10.5, and 11.5. The solutions were placed in a 37˚C water
bath for 15 minutes then absorbances were read. While there was little difference in
absorbance and, therefore, specific enzyme activity between varying pH, Figure 3 illustrates a
gradual increase in specific enzyme activity. Ultimately, the highest specific enzyme activity of
crop/gizzard tissue was observed at pH 11.5. The Bradford assay for intestinal pH was
performed using bovine serum albumin (BSA) to calculate a standard curve. An average
absorbance reading of an enzyme extract was applied to the equation of the standard curve to
determine the concentration of total protein in intestine extract was 5.451 mg/ml. To perform
the pH enzyme assay, intestine tissue was added to pNPP at slightly different pH (7.5, 9.5, 10.5,
and 11.5). Once again, absorbances were read to indicate the results shown in Figure
4. Specific enzyme activity increased with pH and was the highest at pH 10.5 then dropped
slightly at pH 11.5.
After determining the optimal pH for the specific activity of ALP, temperatures were
varied to gain insight into enzyme kinetics of L. terristris. Intestines were added to a buffer of
pH 10.5 and pNPP. Solutions were then placed in water baths of various temperatures. These

3. temperatures were 22˚C, 37˚C, 56˚C, 70˚C, and 85˚C. Figure 5 demonstrates the specific
activity of ALP at the varying temperatures. The specific activity of ALP gradually increased
until 56˚C where it peaked then began to decrease with increasing temperatures.
Alkaline phosphatase requires a cofactor for efficient activity. 50mM MgCl2 was added to
pNPP, a pH 10.5 buffer, and intestine tissue to see if the presence of a cation cofactor
increased the activity of ALP. A control solution with 0mM MgCl2 was also ran. Both solutions
showed significant amounts of specific activity but there was a slight increase in the specific
activity in the presence of the cofactor, MgCl2, as seen in Figure 6.
Discussion
Alkaline phosphatase was heavily present in crop/gizzard and intestine
tissues. Mammals, such as humans, do not have a crop/gizzard but the findings of ALP in the
intestine coincide with the location of mammalian ALP. While this information was useful,
histochemical stain does not show the specific activity of ALP present in the intestine
tissue. This led to further examination by means of multiple enzyme assays. The first pNPP
assay that tested the activity of ALP in all four tissues confirmed the highest activity in
crop/gizzard and intestine. Looking back at Figure 1, these results were anticipated[T4]
. Unexpectedly, esophagus and pharynx which seemed to have little to no ALP present during
staining showed some significant levels of ALP activity. Mammalian ALP is not found in the
esophagus which is evidence that L. terristris may not be an excellent model organism for the
drug in question.
The next point of focus was to explore effects of various biochemical test. As previously
mentioned, Bradford assays were conducted to determine protein concentration of all tissues
used in the biochemical tests. This assay allowed for comparisons to be drawn across multiple
tissues and to ultimately determine the characteristics of worm ALP. First, the effects of pH
were analyzed on the tissues where enzyme activity was found to be the highest. It was
established that the optimal pH of crop/gizzard was 11.5. However, crop/gizzard is not found in

4. mammals and, therefore, is not of significance for this assignment. More importantly, the
optimal pH of intestinal ALP was shown to be 10.5. Alkaline phosphatases are characterized by
their ability to perform most efficiently at pH 10.5 so the findings of this experiment confirm
alkaline properties of L. terristris ALP . This was also similar to the maximum activity of ALP in
mammals. However the optimal pH for mammalian ALP activity was determined to be between
8.4 and 10.
Another important biochemical factor of mammalian ALP to consider was enzyme
kinetics. Using water baths of varying temperatures, 56 ˚C was shown to be the maximal
temperature for specific enzyme activity. Higher temperatures showed decreased specific
enzyme activity. This may be due to denaturation of the enzyme. In humans, ALP specific
activity is optimally expressed at 42-45 ˚C and denatured at 56 ˚C. This is further evidence that
biochemical properties of L. terristris ALP may differ from mammalian ALP. Perhaps a more
beneficial study would have included a water bath around 44 ˚C to better assess the kinetics of
worm ALP in comparison to human ALP.
The presence of cofactors may increase mammalian ALP activity. Consequently,
increased specific activity of ALP due to MgCl2 in L. terristris is evidence of corresponding
biochemical characteristics of mammalian ALP. Further studies should be performed to show if
EDTA can inhibit ALP in L. terristris, as it does in mammals.
Overall, based on this study, L. terristris would not serve as a sufficient model
organism. Presence of ALP in the esophagus, optimal pH of 10.5 and higher, differences in
heat stability, and minimal effects of cofactor are all examples of why L. terristris ALP is different
from mammalian ALP.

5. Figure 1a Cross section of Crop/Gizzard of the L. terristris stained with napthol AS-MX-fast
blue at 4x magnification. ALP can be found on the epithelium lining the Gizzard lumen.
Figure 1b. Cross section of intestine of the L. terristris stained with napthol AS-MX-fast blue at
4x magnification. As seen there ALP is located in the epithelium layer of the coelom and lining
the typhlosole.

6. Figure 1c. Cross section of Intestine of the L. terristris stained with napthol AS-MX-fast blue at
40x magnification. As seen there is ALP located within the intestine on the epithelium.
Figure 1d. Cross section of Esophagus of the L. terristris stained with napthol AS-MX-fast blue
at 4x magnification. As seen there is little ALP located in the esophagus.

7. Figure 1e is of the Pharynx of the L. terristris at 4x magnification power. The labeling is for the
places that were recognized on the cross section that was taken. As shown there is very little
detection of ALP in the Pharynx.
Figure 2. Simple activity of ALP in the Tissues that were received from the worm. The crop
gizzard of the L. terristris showed the highest simple activity would mean the that they had the
highest ALP activity.
0
0.001
0.002
0.003
0.004
0.005
0.006
Esophagus Pharynx Intestine C/G
Simpleactivitymicromoleactivity/min
Tissue Type

8. Figure 3. Specific activity in L. terristris Gizzards at different pH where all numbers are 10-6.
The crop/gizzard tissue was tested for specific activity at all different pH to find what pH would
be essential for ALP activity. All other activites to determine activity was held constant. It was
determined that pH 11.5 had the highest specific activity for ALP in the L. terristris.
Figure 4.Specific Activity for ALP in the L. terristris intestines. All numbers were 10-5. The pH
for the ALP activity was changed, but all other test for the ALP activity was held constant so it
would be determined if pH would play a significance in ALP specific activity in a certain tissue.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
8.5 9.5 10.5 11.5
Specificactivity(umolespNP/min/mg)
(10-6)
pH
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
5.5 7.5 9.5 10.5 11.5
SpecificActivity(umolespNP/min/mg)
(10-5)
pH

9. Figure 5. Specific activity for ALP in L. terristris intestines. The tubes of protein were run at
different temperature to see if that would change the specific activity for ALP, but all other steps
were held constant to just isolate for differences in temperature.
Figure 6. Specific activity umoles pNP/min/mg in the L. terristris intestine at a different co factor
molarity. The cofactor was MgCl2 and it was changed from 0 mM to 50mM to see at which ALP
activity would be higher. For this experiment all other steps were held constant to make sure it
would be testing for a change in Cofactor.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
22 37 56 70 85
SpecificActivity(umolespNP/min/mg)
Assay Temp ( C)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
OmM 50mM
SpecificActivity(umolespNP/min/mg)
MgCl2 Buffer

10. Work Citied
Howard, D.R., J. Miskowski, A. Sanderfoot, and R. Redman. “Manual for BIO 315. Lab
Handbook. University of Wisconsin La Crosse. 2016. Print.