NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Evidence for the proximity of a cysteinyl and a tyrosyl residue in the active site of 6-phosphogluconate dehydrogenase

The treatment of 6-phosphogluconate dehydrogenase from Candida utilis with dansyl chloride causes the modification of one amino acid residue per enzyme subunit and the inactivation of the enzyme. Either a cysteine or a tyrosine residue can be modified, depending on the pH of the reaction mixture. The dansyl residue can be transferred from one residue to the other suggesting that the two amino acid residues are close in the tridimensional structure of the active site of the enzyme.     

The effects of nonsuppressible insulin-like protein (NSILP) on cyclic nucleotide metabolism in rat liver.

Nonsuppressible insulin-like protein (NSILP), 100 ng/ml, inhibited cyclic AMP accumulation in rat liver, as stimulated by glucagon, 10^?7M, from 493 ± 12 to 183 ± 7 pmoles/gm tissue (p<0.001), but did not alter basal levels of cyclic AMP, 143 ± 2 pmoles/gm tissue. NSILP, 100 ng/ml, also inhibited cyclic AMP accumulation, stimulated by epinephrine, 5 × 10^?4M, from 387 ± 12 to 233 ± 9 pmoles/gm tissue. With 1 μM as substrate, NSILP, 100 ng/ml, increased cAMP-dependent phosphodiesterase activity in liver slices from 19.08 ± 0.18 to 24.94 ± 0.38 pmoles cAMP hydrolyzed/mg protein/min (p<0.001), but did not alter this enzyme activity in broken cell preparations of rat liver. Cyclic GMP levels in liver slices, 22.5 ± 0.3 pmoles/gm tissue, were increased by NSILP to 36.3 ± 0.5 pmoles/gm tissue (p<0.01). NSILP had no effect on adenylate cyclase activity. These changes, caused by NSILP in cyclic nucleotide metabolism in liver, resemble those described for insulin, and suggest that alterations in cyclic nucleotide levels in liver may be relevant to other hepatic effects of NSILP.     

Antimetabolic activities of 2-fluoro-L-histidine.

2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by ³H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.     

Na+,K+-ATPase: on the nature of the "cross-linked" subunits obtained in the presence of o-phenanthroline and cupric ion.

In previous studies on the quaternary structure of Na⁺,K⁺-ATPase, cupric-phenanthroline complex (CP) has been used for the cross-linking of the enzyme subunits. Here we show that the same products obtained in the presence of CP (α,α-dimer, α,β-dimer, and products of higher molecular weight) are also obtained when the enzyme is exposed to Cu²⁺ without o-phenanthroline. The α,β-dimer (but not the α,α-dimer) is dissociated in the presence of EDTA; indicating that this product is not the result of the covalent cross-linking of the subunits through a disulfide bond. The nature of the α,α-dimer remains to be determined. The findings suggest that the results of “cross-linking” experiments with CP should be interpreted with caution until the products are more clearly identified.     

Biosynthesis of a ubiouitin-related peptide in rat brain and in human and mouse pituitary tumors

A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH₂ terminal amino acid sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after a protein segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalmus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatography this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems.