Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2.

A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.

Nerve growth factor stimulation of mouse embryonal carcinoma cell migration.

Embryonal carcinoma cells localize to a specific array of target tissues including the male submaxillary gland following intracardiac injection. These target tissues or conditioned medium derived from them have been shown to stimulate the in vitro migration of embryonal carcinoma cells. Here we show that in vitro migration of mouse embryonal carcinoma cells is induced by the simultaneous presence of two different components. One of these is a specific requirement for a fibronectin substrate. The other component is present in male submaxillary conditioned medium and may be nerve growth factor (NGF). Migration of embryonal carcinoma cells on a fibronectin substrate could be induced by purified NGF, but not epidermal growth factor, and submaxillary conditioned medium contained very high levels of nerve outgrowth activity which could be blocked by anti-NGF antibody. Only the high molecular weight 7S NGF complex was active in inducing migration, while the low molecular weight 2.5S NGF inhibited migration. Neither type of NGF or submaxillary conditioned medium stimulated the in vitro growth of embryonal carcinoma cells. 7S NGF thus differentially affects murine embryonal carcinoma cells by inducing cell motility but not cell division. That embryonal carcinoma cells require 7S NGF for their migration in vitro raises the interesting possibility that these cells may respond similarly in vivo, and be stimulated by tissue-specific high molecular weight NGF molecules to migrate or extravasate into the parenchyma of target organs.

Purification and characterization of a rat liver ferroactivator with catalase activity.

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.

Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes.

A study of the effect of varying ionic strength on the glucose-induced quenching of tryptophan fluorescence of hexokinase isoenzymes A(P-I) and B(P-II) was carried out at pH 8.3 and pH 5.5. At p/ 8.3 both isoenzymes gave apparently linear Scatchard-type data plots even with protein concentrations and ionic strengths for which both dimeric and monomeric forms of hexokinase coexist in signiciant amounts. Taking inco account a 1% accuracy in the experimental measurements, we concluded that the intrinsic dissociation constants K(M) and K(D), for the binding of glucose to the monomeric and dimeric forms of HkB, are within a factor of two of each other, i.e. K(D)/K(M) less than or equal to 2. The values of K(M), estimated from the apparent K, were so greatly influenced by ionic strength that it is clear that it is meaningless to compare K(M) and K(D) values measured at different ionic strengths as has been done in the literature. Curvature in the pH 5.5. fluorescence-quenching plots for relatively low ionic strengths demonstrates cooperativity for glucose-binding to the dimer, positive for HkA but negative for HkB. In contrast, the binding is relatively non-cooperative at high ionic strength at this pH. These results were attributed to the well known effect of salt-neutralization of side chain electrical charges on the flexibility and compactness of proteins.