The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Trypanosoma brucei: radioimmunoassay of variant surface glycoproteins from organisms grown in vitro and in vivo

Bloodstream forms of Trypanosoma brucei that were infective for mammals, when grown in vitro at 37 C for 29 days or 25 months had amounts of variant surface glycoprotein similar to the amounts from bloodstream forms isolated from infected rat blood. The amounts were measured by competition radioimmunoassays for both unique and cross-reacting determinants and the results were the same, providing evidence that a single type of variant surface glycoprotein was measured. Neither radioimmunoassay detected determinants of variant surface glycoproteins in trypanosomes transformed by culturing at 27 C to insect forms not infective for mammals.     

Characterization of human blood platelet membrane proteins and glycoproteins by their isoelectric point (pI) and apparent molecular weight using two-dimensional electrophoresis and surface-labelling techniques

Intact human blood platelets were radioactively labelled at the surface by techniques specific for proteins or glycoproteins. Labelled platelet samples were analyzed by a high-resolution two-dimensional separation system involving isoelectric focusing in the first dimension and discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second. The major platelet membrane glycoprotein (GP) bands (Ib, IIb, IIIa and IIIb) were found to be highly heterogeneous even after removal of terminal sialic acid residues. Lactoperoxidase-catalyzed iodination of platelets showed that the major labelled proteins (Ib, IIb, IIIa and IIIb) had altered isoelectric points ($\text{p}\text{I}$) and molecular weights after neuraminidase treatment. A number of membrane glycoproteins previously undetected by one-dimensional gel electrophoresis were demonstrated and good evidence provided that the major platelet surface proteins are glycosylated.     

Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.     

Isolation and characterization of an extracellular hydroxyproline-rich glycoprotein and a mannose-rich polysaccharide from Eudorina californica (Shaw)

Mucilage and colony walls of E. californica were separated from the cells by homogenization, filtration, and differential centrifugation. The chief components of the mucilage were a high-molecular-weight (MW) hydroxyproline-rich glycoprotein and a very high-MW polysaccharide in the proportions 47% and 34%, respectively. The glycoprotein consisted of galactose, arabinose, xylose and an unidentified neutral sugar; and the amino acids cysteine, aspartic acid, glutamic acid, arginine, lysine, glycine, serine, methionine, histidine, alanine, proline, hydroxyproline, tyrosine, threonine, valine, phenylalanine, isoleucine and leucine. The principal sugar of the polysaccharide was mannose. The chemical composition of the colony walls was essentially the same as that of the glycoprotein in the mucilage except that there was almost twice as much hydroxyproline. Also the protein content of the colony walls was 34% while that of the glycoprotein in the mucilage was 22%. No glucose, sugar acids or nucleic acids were found in the extracellular matrix.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.