Hybrid ribosome formation from Escherichia coli and chloroplast ribosome subunits.

A 70S ribosome was prepared from a 30S ribosome subunit from Euglena gracilis chloroplasts and a 50S ribosome subunit from Escherichia coli. This hybrid ribosome was active in polyuridylic acid-directed polyphenylalanine synthesis.

Ribosome-catalyzed polyester formation.

Deamination of phenylalanyl-transfer RNA with nitrous acid yields the alpha-hydroxyacyl analog, phenyllactyl-transfer RNA. When this is incubated in a protein-synthesizing system directed by polyuridylic acid, it yields an acid-precipitable, alkali-labile polyester of phenyllactic acid.

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

Biosynthetic and structural studies on pheomelanin.

13C-NMR spectroscopy of pheomelanin biopolymers, prepared from isotopically enriched precursors, has been developed as a tool for structure elucidation of melanins. By employing large pulse-widths and short cycle time, only the signals originating from labeled carbons are observed in the high-resolution spectra of these polymers.

Tropomyosin from human erythrocyte membrane polymerizes poorly but binds F-actin effectively in the presence and absence of spectrin.

Actin in the human erythrocyte forms short protofilaments which are only long enough to accommodate tropomyosin monomers (Shen, B.W., Josephs, R. and Steck, T.L. (1986) J. Cell Biol. 102, 997-1006). This interaction between actin and tropomyosin monomers is predicted to be weak, since tropomyosin polymerization parallels its affinity for F-actin. We examine the binding of human erythrocyte tropomyosin to actin in the presence and absence of spectrin and its ability to polymerize. The binding of human erythrocyte tropomyosin to F-actin is not affected appreciably by the present of spectrin. Saturating F-actin with erythrocyte tropomyosin, however, weakens the binding of spectrin dimers to actin. Although tropomyosin from human erythrocyte and rabbit cardiac muscle have similar affinity for F-actin, the polymerizability of erythrocyte tropomyosin as determined by viscosity measurements is much reduced relative to muscle tropomyosin. This unusual property of erythrocyte tropomyosin is likely due to differences in its primary structure from other known tropomyosin at the amino and carboxyl terminal regions which are responsible for its head-to-tail polymerization and cooperative binding to F-actin. Analysis of the distribution of tyrosine by 2-dimensional tryptic mapping of 125I-labelled erythrocyte tropomyosin shows that tyrosine at positions 162, 214, 221, 261 and 267 in rabbit cardiac tropomyosin are conserved in human erythrocyte tropomyosin but Tyr-60 is absent. This observation suggests that erythrocyte tropomyosin has a carboxyl terminal region similar to its muscle counterparts but its amino terminal region resembles that of platelet tropomyosin which also lacks Tyr-60.

Immunochemical characterization of the transglutaminase-catalyzed polymer of activated platelets.

Platelet proteins which contribute to transglutaminase-catalyzed polymer formation in activated platelets were identified by an immunochemical approach using the Western blot technique. The cross-linked polymer was purified from thrombin- or Ca2+-ionophore A23187-activated platelets by a sucrose density gradient procedure in reducing conditions and in the presence of a non-ionic detergent. Following transblotting of non-crosslinked platelet proteins from a Laemmli-type gel onto nitrocellulose, the platelet protein "lanes" were incubated with unabsorbed antibodies, or antibodies absorbed with purified polymer. Antibodies to native whole platelets as well as specific antibodies were used. The results show that myosin, actin, glycoproteins IIb, IIIa, and tubulin are present in the polymer.