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 dispennic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.

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.

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Glaeser and others have suggested that low-dose microscopy of thick, crystalline macromolecules might be more advantageously carried out at higher voltages. To investigate this, the HVEM has been constantly improved by redesign and modification of existing components in order to improve its reliability, stability, versatility, and ease of operation. This has been achieved by improvements in the camera, vacuum system, vibration reduction and installation of an image intensified TV camera with digital image memory; online PDP 11/23 computer for real time 2D FFT computations ; minimal exposure system ; digital fine control of focus, push button selection of five magnifications with independent control of C2; and design of a new cold stage for imaging of protein crystals. Further, a set of programs which provide for image analysis and processing, and synthesis of protein structures from electron micrographs of protein crystal arrays developed at Donner Laboratory, UC Berkeley, has now been implemented on a VAX 11/780 computer associated with the Astronony department at Madison.