Cell surface glycosyltransferase activities during normal and mutant (TT) mesenchyme migration

Migrating cells possess surface glycosyltransferase activity toward extracellular substrates, and the appearance of enzyme activity coincides with the onset of cellular migration (Shur, 1977a, Shur, 1977b, Develop. Biol.58, 23–39, 40–55; E. A. Turley and S. Roth, 1979, Cell17, 109–115). In this paper, surface glycosyltransferases were examined during normal and $\text{T}\text{T}$ mutant mesenchyme migration. Of six glycosyltransferases that were assayed, only galactosyltransferase was present at significant levels on the cell surface, despite the presence of a variety of intracellular glycosyltransferases. All controls have been performed to show clearly the enzyme activity was cell surface localized. In both normal and $\text{T}\text{T}$ embryos, surface galactosyltransferase activity was localized, by autoradiography, primarily to migrating mesenchymal cells, and to a lesser degree, to presumptive neural epithelium. During primitive streak formation, putative $\text{T}\text{T}$ embryos were devoid of surface galactosyltransferase activity. However, as development progressed, the $\text{T}\text{T}$ level of activity eventually exceeded wild-type levels by two- to sixfold and was evident in $\text{T}\text{T}$ tissues prior to the onset of microscopic pathology. Other surface enzymes assayed did not show any $\text{T}\text{T}\text{-}\text{dependent}$ increase in activity. The extracellular galactosyl acceptors were not chloroform:methanol soluble, and glycopeptides prepared by exhaustive Pronase digestion were excluded from Sephadex G-50. This large galactosylated glycoconjugate was readily digestable with endo-β-galactosidase, and, therefore, is similar to the poly-N-acetyllactosamine chains previously identified on early embryonic tissues (A. Kapadia, T. Feizi, and M. J. Evans, 1981, Exp. Cell. Res.131, 185–195; T. Muramatsu, G. Gachelin, M. Damonneville, C. Delarbre, and F. Jacob, 1979, Cell18, 183–191; A. Heifetz, W. J. Lennarz, B. Libbus, and Y. -C. Hsu, 1980, Develop. Biol.80, 398–408). These results support an involvement of surface galactosyltransferases in mesenchyme formation and during migration on poly-N-acetyllactosamine substrates.     

Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians

Fertilization of frog eggs by frog sperm is inhibited if the egg's membrane potential is positive (N. L. Cross and R. P. Elinson, 1980, Dev. Biol.75, 187–198); however, fertilization of salamander eggs by salamander sperm does not depend on membrane potential (M. Charbonneau, M. Moreau, B. Picheral, J. P. Vilain, and P. Guerrier, 1983, Dev. Biol.98, 304–318). Since salamander sperm can fertilize frog eggs, we have investigated whether this cross-fertilization is voltage dependent. If, during insemination with Notophthalmus sperm, Xenopus eggs were voltage clamped between +7 and +20 mV, fertilization proceeded in $\text{7}\text{10}$ (70%) of the clamped eggs, compared to $\text{38}\text{48}$ (79%) of the neighboring eggs. In control experiments in which voltage-clamped Xenopus eggs were inseminated with Xenopus sperm, fertilization proceeded in only $\text{1}\text{10}$ (10%) of the clamped eggs, compared to $\text{59}\text{60}$ (98%) of the neighbors. Similar results were obtained with cross-fertilization experiments between Notophthalmus sperm and Rana eggs. These experiments indicate that the voltage dependence of fertilization depends on the species of sperm.     

Large scale isolation and storage of Neurospora plasma membranes.

Functionally inverted plasma membrane vesieles isolated from the eukaryotic microorganism Neurospora crassa generate and maintain a transmembrane electrical potential via ATP hydrolysis catalyzed by a plasma membrane ATPase (G. A. Scarborough, 1976, Proc. Nat. Acad. Sci. USA73, 1485–1488). In order to facilitate investigation of the molecular mechanism of the electrogenic ATPase, and other transport systems, we have developed a method for the large scale isolation and storage of Neurospora plasma membranes in a stable form. Large quantities of open plasma membrane sheets (ghosts) are isolated by a scaled-up modification of the original method (G. A. Scarborough, 1975, J. Biol. Chem.250, 1106–1111) and stored at ?26°C in 60% glycerol ($\text{v}\text{v}$). As needed, the ghosts are washed free of glycerol and then converted to closed vesicles by a modification of the original method. With this technique, plasma membrane vesicles with normal electrogenic pump activity can be prepared daily in approximately 2.5 h.     

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP^2?, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057). It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.     

On the within-sibship variance

An approximate formula for the expected within-sibship genotypic variance of a polygenic, diallelic, additive character is obtained for arbitrary recombination between the loci affecting the character. The formula is exact, when there is no recombination, or when the recombination is free. It is also shown that, if the value of $\text{1}\text{2}\text{V}{}_{\mathrm{k}}$ (one-half of the parental genotypic variance) is assigned to the within-sibship genotypic variance, as in the model of Cavalli-Sforza and Feldman (1976, Proc. Natl. Acad. Sci. USA73, 1689–1692), it implies the assumptions of random mating and of the perfect linkage. If, on the other hand, the value of $\text{1}\text{2}\text{V}{}^0$ (one-half of the linkage equilibrium genotypic variance) is assigned to the within-sibship variance, as in the model of Rice, Cloninger, and Reich (1978, Amer. J. Hum. Genet.30, 618–643), it implies the assumptions of random mating and either of the free recombination, or of the linkage equilibrium, if the recombination is not free.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using "differential dissociation" techniques.

The steroid hormone-receptor complex concentrations measured by “differential dissociation” techniques have to be corrected to obtain the true concentrations of receptor binding sites (B_s). For the calculation of B_s, the parameters kn (product of the equilibrium association constant and the concentration of binding sites of the “nonspecific” component) and f (fraction of the nonspecific binding measured in the experimental estimates of bound ligand by a given technique), previously proposed by Blondeau and Robel (J. P. Blondeau and P. Robel, 1975, Eur. J. Biochem.55, 375–384) are important. A new parameter of interest, ? [$\text{?}\text{=}\text{knf}\text{(kn + 1)}$], is discussed. The measurement of this parameter ? for three “differential dissociation” techniques allows the comparison of their efficiency and their reliability under various conditions for hormone receptor measurement in cytosol. Charcoal and hydroxylapatite methods are more efficient than the Sephadex G-25 filtration method. It is demonstrated that the “isotopic dilution” correction generally used for the estimation of the background of a given technique may be incorrect whatever the method of correction. A new method, the “double concentration measurement,” is developed. This method is simple, rapid, and precise. It requires two receptor binding measurements at two different saturating concentrations of ligand. This method allows the measurement of the estradiol receptor binding activity from calf uterine cytosol, with an error of less than 5% in samples containing the receptor either free or previously complexed with radioactive hormone, even in the presence of very high concentrations (≤0.5 μm) of radioactive steroid.