Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

Antibody development against northern fowl mites (Acari: Macronyssidae) in chickens.

This study reports the development of an antibody against protein(s) from the tissue of the northern fowl mite, Ornithonyssus sylviarum (Canestrini & Fanzago). Northern fowl mite proteins were obtained by affinity chromatography and used for immunization. Western blot analysis identified proteins that were reactive with sera from birds immunized with the antigen; this indicated that serum antibodies against the northern fowl mite had been produced. Chickens that had been immunized or infested, or both, with the northern fowl mite produced sera that were reactive with a 100 kilodalton (kD) protein. The response was greater if the chicken had been immunized with the antigen and infested with the northern fowl mite. Experimentally immunized and infested chickens experienced limited decreases in the levels of northern fowl mite infestation. Survival of bloodfed mites after ingestion of the immune chicken blood was assessed in an in vitro feeding study using blood-filled parafilm sacs; minor differences in northern fowl mite feeding tendencies were noted. The chickens developed antibodies to the northern fowl mite proteins, but this immunity did not decrease the infestation level or in vitro feeding.

Partial purification of a receptor for the adipokinetic hormones from Musca autumnalis face flies.

Partial purification of the receptors for the neurohormones, diptera corpora cardiaca factors 1 and 2 (DCC1 and DCC2) was achieved. Receptor proteins were obtained from the abdomens of face fly, Musca autumnalis De Geer. Purification methods included detergent solubilization, affinity chromatography, and polyacrylamide gel electrophoresis. Analysis by gel electrophoresis has identified two proteins from this partial purification with relative molecular weights of 45 and 90 kD. A crude receptor preparation was used to develop a ligand binding assay with radiolabeled (tritiated and iodinated) DCC1. Ligand binding was inhibited by 90% when excess unlabeled DCC1 was added to the assay mixture. Ligand binding was optimum at pH 7.5. Binding saturation occurred at approximately 12 picomole radiolabeled ligand concentration. Because DCC1 and DCC2 have been shown to effect the lipid and trehalose levels in the insect an understanding of the neuropeptide-receptor interaction is important for the development of new methods of control of dairy and poultry muscoid flies.