The significance of breast milk biotinidase.

Biotinidase (EC 3.5.1.12) activity in human breast milk was first demonstrated by us, using a newly developed method. The enzyme activity was relatively low compared with that in human serum. However, its activity increased significantly with thiol-reactive agents. Taurine and glutathione also enhanced the enzyme activity considerably. From these results, we speculate that biotinidase in milk would be protected from various inactivation-mechanisms in the same manner as milk lipase. Biotinidase might play an important role in the intestinal absorption of the indispensable vitamin, biotin, during infancy, when enzymes in the pancreatic juice are still immature.

Phosphorylation of rat brain calmodulin in vivo and in vitro.

After injection of [32p]orthophosphate into the third ventricle of rat brain, calmodulin(CaM) was prepared from soluble(S2) and particulate(P2) fractions of the whole brain and analyzed by SDS-PAGE in the presence or absence of Ca2+ followed by autoradiography. CaM from both fractions(S2 and P2) was significantly phosphorylated by endogenous protein kinase(s) of rat brain. The incorporation of radioactive phosphate into membrane-bound CaM from the P2 fraction was much higher than that of soluble CaM from the S2 fraction. CaM was phosphorylated in vitro by casein kinase 2 but not by casein kinase 1 or by cyclic AMP-dependent protein kinase, suggesting that casein kinase 2 may be, at least in part, responsible for the phosphorylation of CaM even in vivo.

Effect of glucose and pyruvate metabolism on membrane potential in synaptosomes.

Fluorescence changes of rhodamine 6G in synaptosomal suspension, which are correlated to changes in membrane potential in synaptosomes, were measured in the presence of various monosaccharides and organic acids. Addition of D-glucose, D-mannose, pyruvate and L-lactate hyperpolarized the membrane potential, whereas D-fructose, L-glucose, D-galactose, citrate, succinate and L-glutamate were without effect on the membrane potential. Hyperpolarization induced by D-glucose was inhibited by cytochalasin B, phloretin, iodoacetate, F- and 2-deoxy-D-glucose, but not inhibited by oligomycin or phlorizin. On the other hand, hyperpolarization induced by pyruvate was inhibited by alpha-cyanocinnamate or phloretin, but not inhibited by cytochalasin B or F-. Elimination of Na+ in physiological saline depressed hyperpolarization of membrane potential induced by addition of D-glucose, L-lactate or pyruvate. These results suggest that the activity of (Na+ + K+)-ATPase in plasma membranes of synaptosomes is increased by ATP formed by glycolysis, and that the accumulated K+ in synaptosomes hyperpolarizes the membrane potential.

Fluorescence changes of rhodamine 6G associated with changes in membrane potential in synaptosomes.

The intensity of rhodamine 6G fluorescence was found to be a useful scale for measuring the membrane potential in synaptosomes. The fluorescence of rhodamine 6G in synaptosomal suspensions increases with depolarization in the synaptosomes induced by the replacement of cations in the medium or by the addition of agents known to depolarize the membrane potential. Considering the character of the dye, we have derived an equation which gives the relation between the fluorescence intensity of the dye and the membrane potential. The change in membrane potential (diffusion potential) of synaptosomes was calculated using the equation. The calculated membrane potential was proportional to the logarithm of the K+ concentration above 20 mM, and the slope of membrane potential against log [K+] was about 52 mV per decade of concentration. The permeability ratio (Px/Pk; the ratio of the permeability constants of a given cation, X+, and K+) was estimated from the calculated membrane potential.