Lynwood R. Yarbrough

Synthesis and properties of a new fluorescent analog of ATP: Adenosine-5′-triphosphoro-γ-1-(5-sulfonic acid) napthylamidate

Abstract An analog of ATP has been synthesized which contains the fluorophore, 1-aminonapthalene-5-sulfonate attached via a γ-phosphoamidate bond. This analog is strongly fluorescent (quantum yield = 0.63) with an emission maximum at 460 nm; the excited state lifetime is 20 nsec. It is a substrate for DNA-dependent RNA polymerase of E. coli and wheat germ RNA polymerase II. It is also a substrate for E. coli valyl t-RNA synthetase, venom phosphodiesterase, and potato apyrase. Cleavage of the α-β phosphoryl bond as a result of RNA synthesis or by venom phosphodiesterase produces a 15 nm red shift in the fluorescence emission spectrum. This property should make this nucleotide useful for studies of the mechanisms of enzymatic reactions involving cleavage of the α-β phosphoryl bond.

Refolding and release of tubulins by a functional immobilized groEL column.

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.

Effects of age on myosin and creatine kinase isoforms in left ventricles of fischer 344 rats

Left ventricles of hearts from male Fischer 344 rats of 2, 8 and 23 months of age were analyzed to determine if aging results in significant alterations in the isoform distribution of myosin and creatine kinase protein and mRNAs. Left ventricles of maturing (2-month) rats contained almost exclusively alpha-myosin heavy chain (MHC) mRNA and protein. In adults (8 months) there was a 5-fold increase in beta-MHC (fetal isoform) and an approximate 10% decrease in alpha-MHC mRNA levels, relative to 2 months. By 23 months (senescence), beta-MHC mRNA levels had increased by 11-fold and alpha-MHC mRNA levels had decreased by about 30%. These changes corresponded to an increase in the relative proportion of beta-MHC protein, from undetectable levels at 2 months, to about 40% by 8 months and to about 60% by 23 months. Increased levels of beta-MHC and its mRNA in older rats correlated with decreased serum thyroid hormone levels. The specific activity of creatine kinase in crude homogenates decreased with age, as has been reported previously. Relative to 2-month controls, the specific activity of creatine kinase had decreased by 21% at 8 months and by 37% at 23 months. Analysis of creatine kinase activity showed no large increase in levels of the fetal (B) isoform with age, as was found for myosin. Levels of mRNAs encoding the B and M isoforms of creatine kinase were significantly reduced in senescent rats. Thus, the decreased levels of creatine kinase in aging rats is correlated with decreased levels of mRNA encoding the BCK and MCK isoforms but not an isoform shift.

Both isoforms of mammalian phosphatidylinositol transfer protein are capable of binding and transporting sphingomyelin

The structurally related mammalian alpha and beta isoforms of phosphatidylinositol (PtdIns) transfer protein (PITP) bind reversibly a single phospholipid molecule, preferably PtdIns or phosphatidylcholine (PtdCho), and transport that lipid between membrane surfaces. PITPbeta, but not PITPalpha, is reported extensively in the scientific literature to exhibit the additional capacity to bind and transport sphingomyelin (CerPCho). We undertook a detailed investigation of the lipid binding and transfer specificity of the soluble mammalian PITP isoforms. We employed a variety of donor and acceptor membrane lipid compositions to determine the sensitivity of recombinant rat PITPalpha and PITPbeta isoforms toward PtdIns, PtdCho, CerPCho, and phosphatidate (PtdOH). Results indicated often striking differences in protein-phospholipid and protein-membrane interactions. We demonstrated unequivocally that both isoforms were capable of binding and transferring CerPCho; we confirmed that the beta isoform was the more active. The order of transfer specific activity was similar for both isoforms: PtdIns>PtdCho>CerPCho>>PtdOH. Independently, we verified the binding of CerPCho to both isoforms by showing an increase in holoprotein isoelectric point following the exchange of protein-bound phosphatidylglycerol for membrane-associated CerPCho. We conclude that PITPalpha and PITPbeta are able to bind and transport glycero- and sphingophospholipids.

Expression of the alpha and beta tubulin genes of the African trypanosome in Escherichia coli

The African trypanosome, Trypanosoma brucei, contains multiple genes for both alpha- and beta-tubulins, which code for similar if not identical proteins. Studies of the structure and function of trypanosome microtubules have been limited due to the difficulties in obtaining sufficient amounts of purified tubulin. To produce large amounts of purified tubulin for studies of structure and function and to begin developing a system for producing systematic alterations of tubulin structure we have cloned and expressed the alpha- and beta-tubulin genes of T. brucei in Escherichia coli to produce the unfused proteins. Controlled high-level expression of both alpha- and beta-tubulin was achieved using a plasmid vector, pOTS, in which expression is controlled by phage lambda promoter/operator and a temperature-sensitive lambda repressor. The tubulins produced are insoluble, as has been found for many other proteins expressed to high levels in E. coli; they are readily purified to near homogeneity by chromatography on DEAE-cellulose in 7 M urea. N-terminal analysis of the purified proteins indicates that they are initiated correctly and that the N-formyl group is removed from the initiating methionine. This factor will probably prove important in the reconstitution of biologically active tubulin.

Cloning and characterization of nitrogenase genes from Anabaena variabilis

Three cloned Klebsiella pneumoniae nitrogenase gene probes were used to perform genomic Southern hybridization analysis and to screen an Anabaena variabilis-Charon 30 genetic library for Anabaena nitrogenase genes. One of ten phage clones that were isolated using a Klebsiella nifHD gene probe was studied in detail. This clone (ABN 10) contains a 9 kb Eco R1 fragment which was observed in genomic Southerns. ABN 10 does not hybridize with a Klebsiella nifK gene probe. A 2.8 kb Hind III fragment that lies within the 9 kb Eco R1 fragment contains the linked Anabaena nifH and nifD genes; the nifK gene lies at least 4 kb away. Partial sequencing of the nifH and nifD genes confirmed their identity and showed strong conservation of amino acid sequence in comparison to Anabaena 7120, Klebsiella pneumoniae, Azotobacter vinlandii, and Clostridium pasteurianum. When the cloned Anabaena nif genes were put into Klebsiella cells on plasmids, they were not expressed, suggesting that the Anabaena promoters are not recognized by Klebsiella transcription factors.

Comparison of myosin and creatine kinase isoforms in left ventricles of young and senescent Fischer 344 rats after treatment with triiodothyronine.

The effects of perturbation of thyroid hormone levels on expression of myosin and creatine kinase isoforms were examined in maturing and senescent rats. Whereas LV of maturing rats contain only alpha-MHC, LV of senescent rats contain nearly equal amounts of both alpha-MHC and beta-MHC. When maturing rats were made hypothyroid by treatment for 14 days with the antithyroid agent, propylthiouracil (PTU), beta-MHC and beta-MHC mRNA levels increased significantly. Administration of T3 to senescent rats, or maturing rats made hypothyroid by PTU treatment produced similar decreases in levels of both beta-MHC and beta-MHC mRNA. In contrast, treatment with T3 produced little change in creatine kinase isoform distribution. Thus, thyroid hormone appears to play a critical role in regulating expression of the isoforms of myosin but not of creatine kinase.

Expression of chick and yeast β-tubulin-encoding genes in insect cells

Abstract A chick cDNA encoding the β2 isotype of tubulin (β2Tub) was cloned into a baculovirus expression vector designed to produce unfused proteins, and several recombinant viruses (re-viruses) were isolated. Immunoblotting studies of homogenates of insect cells infected with re-virus showed a 50-kDa protein that reacted with antibodies specific for βTub. Cells infected with the re-virus appeared to contain much higher levels of βTub than uninfected control cells, perhaps as much as five- to tenfold higher. Isotype-specific antibody for β2Tub showed little reaction in uninfected cells or cells infected with wild-type virus; strong reaction was found with cells infected with re-virus. Analysis by gel filtration of extracts of cells infected with re-virus showed that almost all βTub eluted in the column void volume, suggesting that it was aggregated or associated with other cell proteins. Recombinant baculoviruses producing Saccharomyces cerevisiae βTub were also isolated. Immunoblotting studies using antibodies specific for yeast βTub showed a 50-kDa protein which was absent in uninfected cells or cells infected with wt virus. Immunofluorescence studies suggest that yeast βTub is incorporated poorly, if at all, into the insect cell cytoskeleton.

X-ray analysis of crystals of rat phosphatidylinositol-transfer protein with bound phosphatidylcholine.

Phosphatidylinositol-transfer protein (PITP) is a soluble, ubiquitously expressed, highly conserved protein encoded by two genes in humans, rodents and other mammals. A cDNA encoding the alpha isoform of the rat gene was expressed to high levels in Escherichia coli, the protein purified and the homogeneous protein used for crystallization studies. Crystals of rat PITP-alpha were obtained by vapor-diffusion techniques using the sitting-drop method. Crystals grow within two weeks by vapor-diffusion techniques in the presence of polyethylene glycol 4000. Both crystal forms pack in the monoclinic space group P21. Crystal form I has unit-cell parameters a = 44.75, b = 74.25, c = 48.32 A and beta = 114.14 degrees. Unit-cell parameters for crystal form II are a = 47.86, b = 73.59, c = 80.49 A and beta = 98.54 degrees. Crystal form I has a Vm of 2.295 A3 Da-1 and an estimated solvent content of 46.4% with one molecule per asymmetric unit, while crystal form II has a Vm of 2.196 A3 Da-1 and an estimated solvent content of 44.0%, assuming two molecules per asymmetric unit.