Alkaline phosphatase of Drosophila melanogaster. I. Partial purification and characterization

Two allelic forms (Aph-4 and Aph-6) of the alkaline phosphatase of late third instar larvae of Drosophila melanogaster have been partially purified and characterized. Both forms of the enzyme are inhibited by inorganic phosphate, cyanide ion, and cysteine. Aph-6 has a pH optimum of 8.0 and Aph-4 of 8.5. Zn²⁺ has been shown to be required for activity. Of the 22 phosphorylated compounds tested as substrates, only O-phosphotyrosine possesses a relative activity equal to that of the artificial substrate, p-nitrophenyl-phosphate. O-phosphothreonine, O-phosphoserine, and phosphoethanolamine are also good substrates. A majority of the compounds exhibits a relative activity of 0.5 or less.Paper No. 3446 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. This study was supported by Atomic Energy Commission Contract AT-(40-1)-3980.     

Partial purification and characterization of locust allatotropin I

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse-phase high-performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2-propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC-equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one-tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.     

Partial purification of plant NAD kinase by calmodulin-Sepharose affinity chromatography

Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant (zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSo₄ polyacrylamide gel electrophoresis of the proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared.