Identification and characterization of a pro-tumor necrosis factor-alpha-processing enzyme from the ADAM family of zinc metalloproteases.

Tumor necrosis factor-alpha (TNF) is initially expressed as a 26-kDa membrane-bound precusor protein (pro-TNF) that is shed proteolytically from the cell surface, releasing soluble 17-kDa TNF. We have identified human ADAM 10 (HuAD10) from THP-1 membrane extracts as a metalloprotease that specifically clips a peptide substrate spanning the authentic cleavage site between Ala76 and Val77 in pro-TNF. To confirm that HuAD10 has TNF processing activity, we cloned, expressed, and purified an active, truncated form of HuAD10. Characterization of recombinant HuAD10 (rHuAD10) suggests that this enzyme has many of the properties (i.e. substrate specificity, metalloprotease activity, cellular location) expected for a physiologically relevant TNF-processing enzyme.

DNA functional groups required for formation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter. Identification via base analog substitutions.

Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed. Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition. Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role. Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit.

We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: interaction of the catalytic subunit and holoenzyme with lin-benzoadenine nucleotides.

The interaction of lin-benzoadenosine di- and triphosphates with the catalytic subunit and type II holoenzymes of adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has been investigated by steady-state kinetics and fluorescence spectroscopy. lin-Benzo-ADP is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (8.0 microM) similar to the Ki for ADP (9.0 microM). This value agrees well with the Kd (9.0 microM) determined by fluorescence polarization titration. Type II holoenzymes from bovine brain and skeletal muscle have Kd values for lin-benzo-ADP of 3.4 microM and 3.5 microM, respectively, and each binds approximately 2 mol/mol of R2C2 tetramer. Furthermore, fluorescence polarization studies indicate that both the catalytic subunit and type II holoenzyme bind lin-benzo-ADP rigidly, so that there is little or no rotation of the lin-benzoadenine portion of the molecule within the nucleotide binding site. lin-Benzo-ATP is a substrate for the phosphotransferase activities of protein kinase with peptides, water, or type II regulatory subunit as phosphoryl acceptors. With Leu-Arg-Arg-Ala-Ser-Leu-Gly as phosphoryl acceptor, the Km for lin-benzo-ATP is 11.3 microM, and that for ATP is 11.9 microM. The Vmax with lin-benzo-ATP is 20% of the Vmax with ATP as the substrate [24.9 +/- 1.8 mumol/(min . mg) vs. 5.0 +/- 1.2 mumol/(min . mg)]. Thus lin-benzo-ATP is the best nucleotide substrate (besides ATP) for the catalytic subunit reported. 1,N6-Etheno-ATP (epsilon ATP), on the other hand, is a poor substrate for the catalytic subunit with a Km of 1.8 mM and a Vmax that is 4% of the Vmax for ATP, making it unsuitable as a fluorescence probe for cAMP-dependent protein kinase.

Synthesis and biological activity of a profluorescent analogue of coenzyme B12.

We describe here the synthesis and chemical properties of linear(lin)-benzoadenosylcobalamin, a coenzyme B12 analogue that has a laterally extended nucleoside in the upper axial position. It is an effective competitive inhibitor of ribonucleotide reductase from Lactobacillus leichmannii. lin-Benzoadenosylcobalamin is nonfluorescent in solution but, on homolytic (light) or heterolytic (acid, cyanide) cleavage of the carbon-cobalt bond, forms fluorescent products. In addition, fluorescence is detectable on binding of the coenzyme analogue to ribonucleotide reductase, and the observed fluorescence polarization of the lin-benzoadenosyl moiety indicates that it is bound loosely to the enzyme when the coenzyme is partially dissociated.

beta, gamma-Peroxy analogs of adenosine and guanosine triphosphate: synthesis and biological activity.

Extended analogs of adenosine triphosphate (ATP) and guanosine triphosphate (GTP), in which a peroxide bridge replaces the terminal bridge-oxygen of the triphosphate chain, have been synthesized. The ability of beta, gamma-peroxy-ATP to inhibit or substitute for ATP in representative enzyme systems and that of beta, gamma-peroxy-GTP, for FTP in protein synthesis was tested.

Synthesis and biological activity of the peroxy-ATP analogue, adenylyl 5'-peroxydiphosphate.

An analogue of adenosine 5'-triphosphate, adenylyl 5'-peroxydiphosphate (beta, gamma-peroxy-ATP), in which a peroxide linkage replaces the terminal bridge oxygen of the triphosphate chain, has been synthesized. The chemical and biological properties of beta, gamma-peroxy-ATP in representative enzyme systems demonstrate its potential usefulness as a probe of enzymatic mechanism and structure.

Chemotaxis towards sugars by Bacillus subtilis.

Many sugars and derivatives were tested in the capillary assay for their attraction of Bacillus subtilis. The major attractants were 2-deoxy-D-glucose, D-fructose, gentiobiose, D-glucose, maltose, D-mannitol, D-mannose, N-acetylglucosamine, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, N-acetylmannosamine, alpha-methyl-D-mannoside, D-sorbitol, L-sorbose, sucrose, trehalose and D-xylose. Only glucose chemotaxis was completely constitutive. Competition experiments were carried out to determine the specificities of chemoreceptors. There were 25 instances of no influence of two sugars on each other's taxis, 92 instances of one sugar interfering non-reciprocally with chemotaxis towards another and 49 instances of two sugars reciprocally competing. However, in most of the last instances, other sugars were identified that interfered with chemotaxis towards one member of the pair but not the other. Thus, nearly all sugars and related compounds appear to be detected by their own chemoreceptors, but many secondary interactions exist.

Mycolic acids. A reinvestigation.

Mycolic acids derived from the cell walls of Mycobacterium bovis BCG, Mycobacterium bovis Bovinus I, Mycobacterium smegmatis, and Mycobacterium tuberculosis H37Rv have been fractionated as their p-bromophenacyl esters by a two-step high performance liquid chromatographic procedure: 1) adsorption chromatography on 10-micrometer particle size silica gel, and 2) reverse phase partition chromatography on a 10-micrometer particle size support containing a C18 bonded phase. This procedure has resulted in the isolation of approximately 24 mycolic acids from each bacterium (very likely homologs of various mycolate types) instead of the two to four that have previously been described. The implication of these results on the previously determined structures of these fatty acids is discussed.