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We report on the chemical preparation of 14C-labeled P1, P3-(5'-guanosyl-5''-adenosyl)triphosphate (1) which is the parent compound of 5'-terminal cap structures of most eukaryotic mRNAs. Incubation experiments of cap (1)- and non cap-structured nucleotides as Ap2A (2) as well as their ribose ring opened derivatives (3, 4) with rat liver subcellular fractions show that cap-degrading nucleases being able to unblock 5' termini are present in nuclei but not in mitochondria.

We report on the enzymatic degradation of 14C-labelled 5',5"-linked dinucleoside triphosphates Gp3A, 2-O-mGp3A, 2'dGp3A, 2'3"ddGp3A and m7Gp3A in rat liver nuclei. The 2'-desoxy- and 2',3'-didesoxy compounds are poorer substrates than the other cap-structured dinucleotides investigated.

(Adenine-14C) or (gamma-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl]-dihydroxydiethylether (rroANP) was obtained from ANP by cleavage of the C-2--C-3' bond by sodium periodate oxidation and subsequent borohydride reduction. Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki = 9.3 X 10(-5) M. Experimental rroANP confirmed that an intact adenine base and three anio.nic charges of the phosphate chain are essential for the recognition between ANP-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system.

(2'-5')-oligoadenylates bearing a 5'-terminal triphosphate or a 5'GTP-group inhibit the activity of a dinucleoside triphosphatase in rat liver nuclei thereby protecting mRNA against 5'-exonucleolytic degradation. (2'-5')-oligoadenylates, on the other hand, were known to enhance the activity of an endoribonuclease, RNaseF. Thus a synergistic effect may be assumed in vivo, i.e. cellular metabolism seems to be protected twice in virus-infected cells.

The conformationally restricted adenine nucleotide analogs 8,3'anhydro-8-oxy-9-(beta-D-xylofuranosyl)adenine-5'-O-tri(di)-phosphate (I), and 8,2'-anhydro-8-oxy-9-(beta-D-arabinofuranosyl) adenine-5'-O-tri(di)-phosphate (II), were prepared chemically as their alpha-32P-labelled compounds and compared with syn-structured 8-bromo AT(D)P in mitochondrial adenine nucleotide translocation. The experimental findings demonstrate that the heterocycle-ribose orientation affects the carrier mediated adenine nucleotide transport very strongly, i.e. a non fixed adenine heterocycle in the anti region is prerequisite for the bound nucleotide to induce the transfer action of the adenine nucleotide carrier.

An ADP analogue, i.e. 3H- or 14C-labeled 5'-diphosphate of 6-(beta-D-ribofuranosyl-methyl)-4-pyrimidinamine, was synthesized, the structure of which was deduced from structure-activity studies on the substrate specificity of the nucleotide-binding center of the inner mitochondrial membrane integrated ADP/ATP carrier protein. Bearing only the minimal but substrate-essential recognition structures, minimum structured ADP (msADP) is bound to the cytosol-facing active center and transported by the highly specific carrier system across the inner mitochondrial membrane.

1. P1, P5-Bis-(5'-adenosyl) pentaphosphate (AP5-A) inhibits "soluble" adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10(-5) M, i.e. about two orders of magnitude higher than the inhibitor contants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme. 2. as to be expected Ap5A which is of the "multisubstrate analogue"-type does not affect mitochondrial nucleoside diphosphate kinase. 3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor speicifically bound. 4. Ap5A is not metabolized by rat liver mitochondria.

We report on the chemical preparation of the NAD+ analog P1, P2-(5'-beta-nicotinamideribofuranosyl-3"-adenosyl)-diphosphate (N3"AD+) by applying the 1,1'-carbonyldiimidazole activation method. The importance of how adenosine is linked to the nicotinamide mononucleotide phosphoriboride (NMNPR+) moiety is discussed with respect to the coenzyme activity in the lactate dehydrogenase catalyzed reaction.

A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.

We developed a coupled dual column system with an optional post-column derivatization for on-line sample processing, trace enrichment and analysis of aromatic 1,2-diol and aliphatic cis-diol biomolecules (e.g. catecholamines, ribonucleosides). The fully automated high-performance liquid chromatography analyzer tolerates the direct injection of proteinaceous fluids by use of a unique bonded-phase precolumn material which allows the simultaneous performance of covalent affinity and size-exclusion chromatography.