Worm Breeder's Gazette 9(1): 56

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

An Acid Phosphatase Activity Localized To The Gut of C. elegans

C. Beh and J. McGhee

We have previously described a gut-localized esterase as a 
biochemical marker with which to study the early events in lineage-
specific gene expression (Abstracts #14 and 86 in May, 1985, CSH; also 
Edgar and McGhee, Developmental Biology, in press).  We wanted to find 
a second gut-specific hydrolase with which to compare the esterase 
expression and an acid phosphatase appears to be a good candidate.  
Adult worms are turned into  extrudates  by cutting in the presence of 
2  g/ml of levamisole (see Lewis, et. al., Genetics 95: 905 (1980)), 
fixed in paraformaldehyde and stained with the following mixture:  50  
l of 20 mg/ml of 1-naphthyl phosphate, 100  l of 1 M NaOH, 850  l 0.15 
M sodium acetate to which is added 100  l freshly diazotized 
pararosaniline.  Final staining pH is close to 5.0.  Staining is 
usually rapid, intense and gut-localized.  However, this staining 
pattern shows two important differences from the esterase pattern.  
Whereas esterase staining seems to occur throughout the cytoplasm of 
all gut cells, phosphatase staining appears localized to the edge of 
the gut lumen in the region of the brush border.  (This same pattern 
is found in worms grown axenically and therefore does not result from 
clinging E. coli).  Moreover, phosphatase staining is not found in the 
anterior six cells of the gut (i.e., those cells which derive from the 
anterior daughters of Ea(1/r)a and Ea(1/r)p cells and which show no 
later nuclear divisions).  Since several cells at the posterior end of 
the gut also do not stain, there seems to be some symmetry in the 
phosphatase distribution but we re not yet certain how this correlates 
with their variable nuclear divisions.  In any case, the staining 
pattern indicates that a spatial decision to express or activate the 
phosphatase is made relatively late in intestinal development.
Under the usual staining conditions, the gut phosphatase appears to 
be the major phosphatase found in crude extracts of unsynchronized 
populations.  Stained isoelectric focusing gels show one major band (
pI about 4.9) along with several minor bands.  Unlike the esterase, 
the phosphatase is stable at its pI and gels can be left to stain 
overnight, allowing phosphatase activity to be detected in the 
equivalent of one worm or less.  Preliminary steps in purification are 
consistent with the phosphatase being a membrane protein (i.e., 
activity is enhanced by Triton X-100) and Triton increases the 
apparent Molecular Weight on Sephacryl columns.  Phosphatase activity 
in crude extracts is inhibited by tartrate but not by other 
phosphatase inhibitors such as alloxan, CuSO4, formaldehyde or EDTA.
We will probably follow the same scheme worked out for the gut 
esterase, namely to purify the enzyme as a first step towards gene 
cloning and to induce isoelectric focusing mutants to locate the 
phosphatase gene on the genetic map.