Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Temperature-jump studies on benzeneboronic acid-serine protease interactions. Subtilisin and alpha-chymotrypsin.

The interactions of benzeneboronic acid (BBA) as a transition state analog with subtilisin (EC 3.4.21.4) and with alpha-chymotrypsin (EC 3.4.21.1) were investigated kinetically by the temperature-jump method using pH indicators. For both enzymes, the concentration dependence of the relaxation time was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow, unimolecular process. The possibility of a trigonal-tetrahedral interconversion of BBA at the active site of the enzyme is discussed.     

Elementary processes in the interaction of serine protease with a possible transition state analog. Subtillisin-benzeneboronic acid system.

The interaction of benzeneboronic acid(BBA), a possible transition state analog, with subtilisin BPN' [EC 3.4.21.14] was studied by the temperature-jump method at various pH's, temperatures and in D2O as well as H2O. From analysis of the concentration dependence of the relaxation times, it was suggested that the subtillsin-BBA interactions consist of at least two elementary steps, a fast bimolecular association followed by a slow unimolecular process. Similar concentration dependence was observed at pH 6.1-6.7 at 25degrees. However, in D2O the reciprocal relaxation times generally decreased compared to those in H2O and became concentration-independent below pD 6.5. The relaxation times were influenced considerably by the temperature. From these results, the slow unimolecular process was assigned to the trigonal-tetrahedral interconversion of BBA at the active site of the enzyme.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.     

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD⁺ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.     

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.