A novel extracellular carbohydrase produced by Lipomyces tetrasporus

Summary The ascosporogenous yeast Lipomyces tetrasporus produced an unusual extracellular carbohydrase. It was purified to homogeneity using ammonium sulphate precipitation and DEAE Bio-gel A ion-exchange chromatography. While retaining highest activity on low-molecular-weight saccharides such as maltose and nigerose, it displays considerable activity towards polymeric substrates including soluble starch. It is particularly unusual in that it also hydrolyses dextran and has a very high affinity for this substrate. The enzyme has an exo-lytic mode of action with the only hydrolysis product, glucose, being released in the -anomeric form. Optimum activity occurs at pH 4.5 and at 50°C. It is a glycoprotein, and has an M _r value of 150 000 (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) — 183 000 (fast protein liquid chromatography) and a pI of 6.0. Offprint requests to: C. T. Kelly     

Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population

Summary We describe a simple method for characterizing a frequent polymorphism (that subsitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer.     

Positive effectors of the binding of an active site-directed amino steroid to rabbit cytochrome P-450 3c

The binding of the amino steroid, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), to rabbit liver cytochrome P-450 3c was studied using purified P-450 3c and liver microsomes prepared from rifampicin-treated B/J rabbits. 22-ABC binds to purified cytochrome P-450 3c producing a type II spectral change reflecting the coordination of the amine with the heme iron of the protein. In the absence of allosteric effectors, the binding is characterized by a Ks of 5 microM. In the presence of alpha-naphthoflavone or progesterone, the Ks decreases to 0.8 microM, indicating that these two compounds serve as positive effectors of the binding of 22-ABC to cytochrome P-450 3c. The antibiotic rifampicin induces cytochrome P-450 3c in rabbit liver microsomes, and the benzo(a)pyrene hydroxylase, estradiol 2-hydroxylase, and progesterone 6 beta-hydroxylase activities of these microsomes are stimulated by alpha-naphthoflavone. Moreover, the progesterone 6 beta-hydroxylase activity catalyzed by these microsomes exhibits a dependence on substrate concentration that is consistent with activation of the enzyme by the substrate, progesterone. The magnitude of the type II spectral change elicited by 22-ABC for microsomes prepared from rifampicin-treated B/J rabbits is greater than that observed for microsomes from untreated rabbits. For microsomes from rifampicin-treated rabbits, the apparent binding constant for 22-ABC was decreased 5-fold in the presence of alpha-naphthoflavone. We propose that the effects of alpha-naphthoflavone and progesterone on the binding of 22-ABC to cytochrome P-450 3c mimic the effects of the two positive effectors on the metabolism of substrates by increasing the affinity of the enzyme for substrate.     

Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.     

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Evidence for posttranslational O-glycosylation of fetuin

Fetuin, a major glycoprotein in the serum of fetal calves that contains three N-linked and three O-linked carbohydrate side chains, was found to be synthesized in the liver with an 18 amino acid signal peptide, Met-X-X-X-X-Leu-Leu-X-Cys-Leu-Ala-X-Leu-X-X-Cys-X-X, and to undergo cotranslational N-glycosylation. In order to examine O-glycosylation, fetuin peptidyl-tRNA was purified from liver and analyzed for O-linked carbohydrate by quantitating the released [3H]GalNAcitol produced after beta-elimination in the presence of NaB3H4. Within the limits of the assay, less than 1.3% of the O-linked chains had been initiated. Additionally, rough microsomes were used to program a cell-free protein synthesis system. A radiolabeled fetuin intermediate was isolated by immunoprecipitation and shown to contain N-linked carbohydrate by binding to concanavalin A and by susceptibility to cleavage by endoglycosidase H. However, this fetuin intermediate was not detectably bound (less than 1%) by GalNAc-specific lectins, which were shown to bind asialoagalactofetuin. These results suggest that O-glycosylation of fetuin is a posttranslational event.     

Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pertussis toxin inhibits retinoic acid-induced expression of tissue transglutaminase in macrophages

Retinoic acid rapidly induces the accumulation of a specific enzyme, tissue transglutaminase (EC 2.3.2.13), in mouse macrophages. We have used the induction of tissue transglutaminase to study the regulation of gene expression by retinoic acid. In this study we report that pertussis toxin can inhibit retinoic acid-induced expression of tissue transglutaminase in mouse resident peritoneal macrophages. This inhibition is paralleled by the ADP-ribosylation of 41,000-dalton macrophage membrane protein.     

Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence

We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent ("polymix"), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)