Role of cellular casein kinase II in the function of the phosphoprotein (P) subunit of RNA polymerase of vesicular stomatitis virus.

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1.

Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.

The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precursor expressed in Escherichia coli were compared using circular dichroic (CD) spectral analysis. The spectra obtained for the intact recombinant and viral proteins were indistinguishable, indicating that the backbone configurations directed by the primary amino acid sequences of the proteins were similar or identical. The structure predictions derived from CD were, in general, inconsistent with a model proposing the eight-stranded beta barrel motif found in several RNA viruses.

The effect of chemical modification of basic amino acid residues on the activation and amidolytic activity of Hageman factor (factor XII).

Modification of arginyl residues of Hageman factor by phenylglyoxal hydrate inhibits activation of this clotting factor in a plasma-free system, that is, in the absence of the other constituents of the contact activation system. Activation is also inhibited by alteration of the other two basic amino acid residues present, lysine and histidine. Chemical modification of histidine and arginine residues does not inhibit the amidolytic activity of activated Hageman factor.

Interactions of avian myeloblastosis virus nucleocapsid protein with nucleic acids.

The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet only a minimal biochemical description of NC-nucleic acid interactions is available.

Fluorimetric analysis of recombinant p15 HIV-1 ribonuclease H.

We have exploited the sole tryptophan residue (Trp535) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (p15 RNase H) by fluorescence spectroscopy. Incubation of purified p15 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in Trp535 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.

Involvement of tryptophan(s) at the active site of polyphosphate/ATP glucokinase from Mycobacterium tuberculosis.

The glucokinase (EC 2.7.1.

Comparison of the substrate-binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases.

A steady state kinetic analysis of the avian myeloblastosis virus/Rous sarcoma virus (AMV/RSV) and human immunodeficiency virus Type 1 (HIV-1) retroviral proteases (PRs) was carried out using a series of 40 peptide substrates that are derivatives of the AMV/RSV nucleocapsid-PR cleavage site. These peptides contain single amino acid substitutions in each of the seven positions of the minimum length substrate required by the PR for specific and efficient cleavage. These peptide substrates are distinguished by the individual enzyme subsites of the AMV/RSV and HIV-1 PRs.

A cationic region of the platelet-derived growth factor (PDGF) A-chain (Arg159-Lys160-Lys161) is required for receptor binding and mitogenic activity of the PDGF-AA homodimer.

Platelet-derived growth factor-AA and -BB homodimers and -AB heterodimers bind with high affinity to the platelet-derived growth factor (PDGF) alpha-receptor. Basic polypeptides such as polylysine and protamine sulfate compete with PDGF for receptor binding, suggesting a role for ligand positive charge in the binding interaction. A pentapeptide amino acid sequence with a cationic tripeptide core is perfectly conserved between the A- and B-chains (Val158-Arg159-Lys160-Lys161-Pro162) and was therefore considered as a possible alpha-receptor-binding domain.

Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme.

Polyphosphate [poly(P)n]:D-(+)-glucose-6-phosphotransferase (EC 2.7.1.

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected.

A structural model for human dihydrolipoamide dehydrogenase.

The hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3.

Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components.

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q.

31P nuclear magnetic resonance (NMR) identification of sugar phosphates in isolated rat ovarian follicular granulosa cells and the effects of follicle-stimulating hormone.

Immature female rats (23-30 days old) were implanted subcutaneously with diethylstilbestrol (DES) in silastic capsules. After 48 h their ovaries were removed and the granulosa cells isolated (Foreman et al. (1984) Life Sci.

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid.

The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M.

What is the role of the cys-his motif in retroviral nucleocapsid (NC) proteins?

Retroviruses encode a small, basic nucleocapsid (NC) protein that is found complexed to genomic RNA within the viral particle. The NC protein appears to function not only in a histone-like manner in packaging the RNA into the particle but also in specifically selecting the viral genomic RNA for packaging. A cysteine-histidine (cys-his) region, usually composed of 14 amino acids and reminiscent of the 'zinc fingers' of transcription factors, is the only highly conserved sequence element among the retroviral NC proteins.

Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and 13C NMR.

The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KC1 as a function of pH by 13C NMR spectroscopy.

Molecular biology of the human pyruvate dehydrogenase complex: structural aspects of the E2 and E3 components.

The availability of the primary amino acid sequences of the E2 of PDC, alpha-KGDC and BCKADC from several prokaryotic and eukaryotic species has allowed us to compare the structural aspects of human PDC-E2 with those of the E2 components from the other complexes. The PDC-E2 components from all the species examined so far contain three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. The primary structure of the lipoyl-bearing domain shows considerable variation in its size, ranging from one to three repeating units of approximately 110 amino acids, but essentially preserving its function in the E2 components.

Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers".

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Cys. All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus.

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum.

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR).

Deletion of lysine 13 alters the structure and function of parathyroid hormone.

A peptide of unknown structure was found as a side product in a commercial preparation of the 1-34 fragment of bovine parathyroid hormone (PTH). CNBr cleavage and amino acid analysis showed that this peptide is the des-lys-13 form of 1-34 bovine PTH. The peptide thus represents a deletion mutant of PTH and structure-function studies are of interest.

Proton NMR studies of the biologically active 1-34 fragment of bovine parathyroid hormone: examination of a structural model.

Proton NMR spectra of the biologically active 1-34 fragment of bovine parathyroid hormone (bPTH) were studied as a function of pH over the range of pH 4 to 10, in buffer and in 6 M guanidine DC1. One of the histidine C-2 peaks titrated normally, with a pKa value of 6.8, but the other two histidines in this peptide had pKa values of 6.

Forms of a self associating autoantibody complex between a monoclonal human IgG1 and human serum albumin.

The mode of association of an unusual human autoantibody complex, composed of a monoclonal immunoglobulin, Tu IgG, and human serum albumin was investigated. A crystalline complex forms from these components in the cold and we have shown that it consists of IgG and albumin in a 1:2 molar ratio [Jentoft et al., Biochemistry 21, 289-294 (1982)].

MOLECULAR DESIGNER: an interactive program for the display of protein structure on the IBM-PC.

A BASIC interactive graphics program has been developed for the IBM-PC which utilizes the graphics capabilities of that computer to display and manipulate protein structure from coordinates. Structures may be generated from typed files, or from Brookhaven National Laboratories' Protein Data Bank data tapes. Once displayed, images may be rotated, translated and expanded to any desired size.

Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study.

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up.