Molecular basis for ATP/2,3-bisphosphoglycerate control switch-over (poikilotherm/homeotherm) an intermediate amino-acid sequence in the hemoglobin of the great Indian rhinoceros (Rhinoceros unicornis, Perissodactyla)

The complete primary structure of the two hemoglobin components of the Great Indian Rhinoceros (Rhinoceros unicornis) is presented. The ratio for the two components B(alpha 2 beta I2): A(alpha 2 beta II2) is 6:4. Polypeptide subunits were separated by chromatography on CM-cellulose in a buffer containing 8M urea. The sequence was studied by degradation of the tryptic and hydrolytic cleavage products in a liquid phase sequencer. At position beta NA2 component B has Asp, whereas component A has Glu, an ATP-binding site in fish and reptilian hemoglobins. The other phosphate binding sites i.e. beta NA1 Val, beta EF6 Lys and beta H21 His are identical with 2,3-bisphosphoglycerate-(DPG)binding sites in mammalian hemoglobins, whereby rhinoceros hemoglobin resembles both ATP-sensitive poikilotherm hemoglobin and DPG-sensitive mammalian hemoglobin. The two components (beta I/beta II) additionally differ by exchange of Glu----Gly at position beta A3 and Gln----Lys at position beta GH3. The significance of these changes is discussed. Oxygenation properties of the two hemoglobins components and their dependence on ATP and DPG are given. The structure and function of Rhinoceros hemoglobin may give an insight into the evolution of the organic phosphate binding in vertebrate hemoglobins.     

Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand

Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell-associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.     

Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

The primary structure of hemoglobin from amur-leopard (Panthera pardus orientalis)

The complete amino acid sequence of the major component of hemoglobin from amur-leopard (Panthera pardus orientalis) is presented. The major component accounts for more than 90% of the total hemoglobin. Separation of the globin subunits was achieved by ion-exchange chromatography on CM-cellulose in urea. The sequence was studied by automatic Edman degradation of tryptic and hydrolytic peptides. Alignment was carried out with human hemoglobin sequence. The NH₂ terminus is blocked with Ac-serine. The data are compared with other mammalian hemoglobins and results are discussed with respect to sequence and physiology.85th communication on hemoglobin.     

Isolation purification and properties of a site-specific proteolytic enzyme "valyl-proteinase" from Candida tropicalis

A highly specific proteolytic enzyme cleaving at the carboxyl group of valine has been isolated from Candida tropicalis. Its specificity has been determined by digesting beta-lactoglobulin and a number of synthetic peptides. The enzyme a glycoprotein, has a molecular mass of 40 +/- 7 kDa on the basis of sodium dodecyl sulphate polyacrylamide gel electrophoresis. Its optimum activity occurs at 37 degrees C at a pH between 8-9. It has been named "Valyl-proteinase" because of its selective cleavage.     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm² of growth flask). Results show that the apparent expression of extended Le^a-Le^x, Le^a and Le^x, sialyl Le^a, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.Abbreviations Mab monoclonal antibody - FIT fluorescein isothiocyanate - TACA tumor associated carbohydrate antigen     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.     

A microprocedure for extracting tissue nucleotides for analysis by high-performance liquid chromatography.

A method is described for the preparation of concentrated tissue extracts for nucleotideanalysis by high-performance liquid chromatography (hplc). Ten to one hundred milligrams of tissue was extracted in a combined weighing-homogenizing-centrifuge tube using a trichloracetic acid (TCA)-methanol extractant containing a radioactive internal standard. This extractant eliminated nucleotide interconversion which was found to occur when TCA alone was employed. High ATP/ADP and ATP/AMP ratios were observed and recoveries of greater than 97% were obtained with exogenous radioactive nucleotides. The method has been applied successfully in studies on muscle, heart, liver, kidney, lung, brain, and subcellular fractions.     

Alkalinization of phosphorylase kinase-deficient muscle during tetanic contraction

The intracellular pH of resting and stimulated muscle was monitored by two independent methods: measurement of pH iniodacetate-treated homogenates of freezeclamped tissue and the absorbance at 550–443 nm of intracellular neutral red dye in vivo. During tetanic stimulation, muscle of phosphorylase kinase-deficient mice shows a transient alkalinization whereas muscle in normal mice becomes more acid under similar conditions. The alkalinization appears to be caused by abnormally rapid AMP deamination associated with adaptation to phosphorylase kinase deficiency.