Morphology of the site of sperm entry on the frog egg.

We located the site of sperm entry on the frog (Rana pipiens) egg using light and scanning electron microscopy. The site changed from a small, microvilli-free structure at 20 min after insemination to a clump of elongated microvilli at 40 min, to a distinct patch of microvilli by 2 hr. The sperm entry site responded differentially to hypertonicity. At 40 min, the site was found in a circular depression which was due to the sperm aster and which expanded to a rigid area in the animal half. We consider how these observations may be related to gray crescent formation.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Photoaffinity labeling of the adenosine transport system in adipocyte plasma membranes

A membrane component involved in the transport of adenosine in adipocytes has been identified utilizing the techniques of photoaffinity labeling with the adenosine derivative, 8-azidoadenosine. In the absence of light, adenosine and 8-azidoadenosine exhibited similar transport characteristics. In addition, adenosine was shown to be a competitive inhibitor of 8-azidoadenosine uptake, and the photoprobe, a competitive inhibitor of adenosine uptake. Analysis of the nucleotide metabolites indicated that the photoprobe was metabolized in a similar fashion to that observed for adenosine. Several nucleoside transport inhibitors were also equally effective in inhibiting the uptake of both nucleosides. These results suggest that 8-azidoadenosine is transported by the same membrane system as adenosine. Photolysis of 8-azido[2-³H]adenosine in the presence of adipocytes resulted in the covalent incorporation of the photoprobe into the plasma membrane fraction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that essentially all of the radioactivity was incorporated into a glycoprotein with a molecular weight of 56,000. This labeling was inhibited by greater than 90% when the photolysis was carried out in the presence of excess adenosine or the transport inhibitors, persantin or theophylline. Fractionation of the labeled plasma membranes by dialysis against water (pH 9.5) indicated that approximately 75% of the radioactivity was associated with a glycoprotein which resisted solubilization by this procedure. These results suggest that the major labeled species is a 56,000 M_r intrinsic membrane glycoprotein which may function as a component of a transmembrane assembly involved in the transport of adenosine.     

Classification of DNA polymerase activities from ovaries of the frog,Xenopus laevis

Four distinct DNA polymerase activities were isolated from ovaries of the frog Xenopus laevis. Specific assays for each activity were established. The isolated activities were characterized by molecular weight, template-primer preferences, and sensitivity to specific inhibitors as Xenopus laevis ovarian DNA polymerases-α₁, -α₂, -β, and -γ. All previously described Xenopus laevis DNA polymerases were classified using these properties.     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.     

Comparison of rat and human left ventricle beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Beta adrenergic receptors were identified in rat myocardial left ventricle and human papillary muscle by using the antagonist radioligand 3H-dihydroalprenolol. The number (37.3 and 44.5 fmol/mg of protein, respectively in rat and man), and the KD (1.6 and 2.8 nM, respectively in rat and man) of beta receptors were not significantly different. Adrenergic receptors of both beta 1 and beta 2 subtypes were found to coexist in the left ventricle. The relative proportions of the two beta receptor subtypes were determined by the use of competition radioligand selective binding and computer modelling techniques employing the subtype selective antagonists ICI 118,551 (beta 2 selective) and atenolol (beta 1 selective) in rat or metoprolol (beta 1 selective) in man. The rat left ventricle contained about 74% beta 1 and 26% beta 2 adrenergic receptors, human left ventricle papillary muscles contained about 69% beta 1 and 31% beta 2. Human and rat left ventricles contain both beta 1 and beta 2 adrenergic receptors with similar affinities. Rat might be a model for the study of human myocardial beta adrenergic receptors.     

Radioactive labeling of alpha1-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives.

Human plasma α₁-antitrypsin (α₁-AT), bovine trypsin, and α-chymotrypsin were labeled with either ¹⁴C or ³H by reductive methylation. The labeled inhibitor retained the capacity to inactivate and to form 1:1 molar complexes with either the unlabeled or labeled trypsin and α-chymotrypsin. After intravenous injection of reductively methylated α₁-AT into rats, the labeled glycoprotein showed a circulating half-life of 12 h. When the N-acetylneuraminic acid residues were removed from the labeled α₁-AT by neuraminidase in vitro, injection into rats of this product resulted in a rapid (half-life of about 5 min) and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver of over 75% of the injected dose. The reductively methylated radioactively labeled trypsin and chymotrypsin experienced no loss of enzymatic activities. They showed the ability to form complexes in vivo with the two major plasma inhibitors, namely, α₁-AT and α₂-macroglobulin. High-voltage paper electrophoretic separation of acid hydrolysates of the labeled proteins revealed that ?-N-monomethyllysine and ?N,N-dimethyllysine are the only residues found to be radioactive.     

Photoaffinity labeling of a microtubule-associated cyclic AMP-binding protein

8-Azido cyclic AMP has been used as a photoaffinity probe to identify cyclic AMP-binding proteins in microtubule preparations. Bovine brain microtubule proteins and rabbit muscle protein kinase were incubated with the photoaffinity ligand in reduced light for 15 min, without additions or with 100-fold excess unlabeled cyclic AMP or 5′-AMP. Samples were then irradiated at 254 nm at a distance of 1 cm for 5 min, in ice. After irradiation aliquots were taken for electrophoresis in one or two dimensions. Polypeptides which bound the photoaffinity label were visualized by autoradiography. The apparent molecular weights of the most prominent 8-azido ³²P-cyclic AMP-binding proteins are in the same range as those of the RII of the muscle enzyme. Following two-dimensional electrophoresis the major microtubule-associated cyclic AMP-binding proteins resolve as two spots with about the same pI (~pH 5.0) but slightly different molecular weights. Both spots are in the molecular weight range of the tubulins but they are clearly resolved from the tubulins in the first dimension. Cyclic AMP, but not 5′-AMP blocks the labeling of these proteins. There are low levels of labeling of the tubulins, the high-molecular-weight MAPs and several polypeptides with molecular weights near tubulin but with more basic pI. The photoaffinity probe has demonstrated that the major microtubule-associated cyclic AMP-binding protein of bovine brain is distinct from other RII proteins and from tubulin isomorphs.     

Glycosyl transfers from UDP-sugars to lipids of plant membranes: identification and specificity of the transferases

Microsome preparations extracted from wheat roots or sycamore cell suspensions catalyzed the transfer of sugar from nucleotide-sugars to endogenous lipidic acceptors. The nature of the products biosynthesized from UDP-Glc, GDP-Glc, UDP-Gal, UDP-Xyl or UDP-Arab was examined. Sterylglycosides were obtained from UDP-Gglc, GDP-Glc or UDP-Xyl. Galactosyldiglycerides were synthesized from UDP-Gal. When UDP-Glc or UDP-Gal was used as a substrate, a membrane-bound 4-epimerase interconverted the epimeric nucleotide-sugars, thereby allowing the simultaneous biosynthesis of galactosyldiglycerides and sterylglucosides. The biosynthesis of free and acylated sterylglucosides from UDP-Glc, without interference of other glycosyl transfer reactions, was obtained by the omission of Mg++ ions from the incubation medium. The biosynthesis of galactosyldiglycerides from UDP-Gal without interference of other transfer reactions was obtained when digitonin was added to the incubation medium of sycamore microsomes.     

1,25,26-trihydroxyvitamin D3: isolation, identification, and biological activity

A polar metabolite of vitamin D₃ has been produced in vitro from either 1,25-dihydroxyvitamin D₃ incubated with kidney homogenate from vitamin D-supplemented chickens or from 25,26-dihydroxyvitamin D₃ incubated with vitamin D-deficient chicken kidney homogenate. This compound was isolated in pure form and identified as 1,25,26-trihydroxyvitamin D₃ by ultraviolet absorption spectrophotometry and mass spectrometry. Furthermore, its periodate cleavage product comigrates with synthetic 1α-hydroxy-25-keto-27-norvitamin D₃ on high-performance liquid chromatography. The 1,25,26-trihydroxyvitamin D₃ is 0.1-0.01 as active as 1,25-dihydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone calcium mobilization.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.     

Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.     

Quantitative Cu(I) determination using X-ray absorption edge spectroscopy: oxidation of the reduced binuclear copper site in type 2 depleted Rhus laccase

We report a procedure, through difference comparison of X-ray absorption edge spectra, for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This technique is tested on copper model systems and then used to quantitatively determine that untreated T2D Rhus laccase contains 70 +/- 15% Cu(I). Whereas excess ferricyanide is demonstrated not to alter the Cu(I) content of the untreated T2D, aqueous peroxide and nitrite at pH 6.0 are shown to oxidize the cuprous type 3 site and generate met T2D protein forms.     

Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts

By immunizing mice with cells from embryonic chick motoneuron cultures, an antiserum was produced which recognizes an antigen(s) restricted to cell surfaces of most, or all, neurons. With the use of this antiserum, the appearance of neuron-specific antigenicity in cells of the embryonic spinal cord was examined by indirect immunofluorescence microscopy. The antigen or set of antigens reacting with this antiserum was first detectable in the neural tube of chick embryos at stage 15–16 (V. Hamburger and H. L. Hamilton, 1951, J. Morphol.88, 49–92). In addition to the neuroblasts located in the mantle layer, some mitotic cells as well as some spindle-shaped cells in the germinal layer were antigen positive. Immunofluorescence microscopy combined with autoradiography revealed that none of the antigen-positive cells could be labeled with [³H]thymidine; thus they do not synthesize DNA, and none of the cells in the DNA synthetic phase expressed the antigen(s). As the neuroblasts do not synthesize DNA after they have differentiated from the germinal cells, we believe that the antigen-positive cells are differentiated elements and that the differentiation of membranes specific for neurons begins already before or during the terminal mitosis of cells which will be defined as neuroblasts.