Purification and properties of acid phosphatase from Drosophila virilis

One allelic form(Acph²) of acid phosphatase (EC 3.1.3.2) specified by Acph locus of Drosophila virilis was purified about 1000-fold in 20% yield. The purified preparation was fairly homogeneous as examined by disc and SDS-polyacrylamide gel electrophoresis. The enzyme protein is considered to be a dimer composed of identical or very similar subunits which have a molecular weight of 50,000. The enzyme is also a glycoprotein which contained 20% neutral sugars and a low content of hexosamines. The optimum pH of the enzyme activity was 5.0 and optimum temperature was 53°C. The Acph² enzyme was shown to be relatively heat stable. The purified preparations have a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{p-}\text{nitrophenyl}$ phosphate of 0.2 mM and a broad substrate specificity. The enzyme was inhibited by compounds which inhibit high-molecular weight-acid phosphatase in eukaryotes, localized to lysosomal fractions. The isoelectric point was 9.8. The amino acid analysis revealed a high content of glycine and alanine, but there was no characteristic feature of the composition which accounted for the high isoelectric point.     

Cell surface glycosyltransferase activities during normal and mutant (TT) mesenchyme migration

Migrating cells possess surface glycosyltransferase activity toward extracellular substrates, and the appearance of enzyme activity coincides with the onset of cellular migration (Shur, 1977a, Shur, 1977b, Develop. Biol.58, 23–39, 40–55; E. A. Turley and S. Roth, 1979, Cell17, 109–115). In this paper, surface glycosyltransferases were examined during normal and $\text{T}\text{T}$ mutant mesenchyme migration. Of six glycosyltransferases that were assayed, only galactosyltransferase was present at significant levels on the cell surface, despite the presence of a variety of intracellular glycosyltransferases. All controls have been performed to show clearly the enzyme activity was cell surface localized. In both normal and $\text{T}\text{T}$ embryos, surface galactosyltransferase activity was localized, by autoradiography, primarily to migrating mesenchymal cells, and to a lesser degree, to presumptive neural epithelium. During primitive streak formation, putative $\text{T}\text{T}$ embryos were devoid of surface galactosyltransferase activity. However, as development progressed, the $\text{T}\text{T}$ level of activity eventually exceeded wild-type levels by two- to sixfold and was evident in $\text{T}\text{T}$ tissues prior to the onset of microscopic pathology. Other surface enzymes assayed did not show any $\text{T}\text{T}\text{-}\text{dependent}$ increase in activity. The extracellular galactosyl acceptors were not chloroform:methanol soluble, and glycopeptides prepared by exhaustive Pronase digestion were excluded from Sephadex G-50. This large galactosylated glycoconjugate was readily digestable with endo-β-galactosidase, and, therefore, is similar to the poly-N-acetyllactosamine chains previously identified on early embryonic tissues (A. Kapadia, T. Feizi, and M. J. Evans, 1981, Exp. Cell. Res.131, 185–195; T. Muramatsu, G. Gachelin, M. Damonneville, C. Delarbre, and F. Jacob, 1979, Cell18, 183–191; A. Heifetz, W. J. Lennarz, B. Libbus, and Y. -C. Hsu, 1980, Develop. Biol.80, 398–408). These results support an involvement of surface galactosyltransferases in mesenchyme formation and during migration on poly-N-acetyllactosamine substrates.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Localization at the ultrastructural level of maternality derived enzyme and determination of the time of paternal gene expression for acid phosphatase-1 in Drosophila melanogaster.

Ultrastructural histochemistry instead of acrylamide gel electrophoresis (see R. Yasbin, J. Sawicki, and R. J. MacIntyre, 1978, Develop. Biol. 63, 00-00) is used to determine the time of paternal gene expression for the enzyme acid phosphatase-1 of Drosophila melanogaster in $\text{Acph-1}{}^{\mathrm{n}}\text{Acph-1}{}^{\mathrm{B}}$ embryos in which the null allele is derived from the female parent. Timed embryos were histochemically stained for acid phosphatase activity according to the lead phosphate method of Gomori and were examined at the ultrastructural level. Enzyme activity, resulting from activation of the paternal Acph-1 gene, is detected as early as 5 hr after fertilization. Maternally derived enzyme in $\text{Acph-1}{}^{\mathrm{B}}\text{Acph-1}{}^{\mathrm{B}}$ embryos is found principally in the yolk regions and around invaginations. This suggests that acid phosphatase-1 functions in yolk digestion and in cell movements during early embryogenesis.