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The paper describes studies on the influence of heat impact in reconstituted skim milk on chemical and functional properties of yoghurt products. Reconstituted skim milk was heated for 20 min at 85 degrees C, 90 degrees C, or 95 degrees C. Ropy (producing exopolysaccharides, EPS) or non-ropy strains of S. thermophilus and L. delbrueckii subsp. bulgaricus were used as starter culture for yoghurt manufacture. The studies have shown that the fermentation times decreased with increasing heat impact when the ropy starter culture was used, while they remained to a far extent unchanged if the non-ropy starter culture was applied. The lactic acid contents of the yoghurt products were in the same range when the milk was heated at 85 degrees C or 90 degrees C, while they were different when milk was heated at 95 degrees C. There was a tendency visible that an increase in preheating leads to increased L(+)- and decreased D(-)-lactic acid contents if the non-ropy culture was applied. Using the ropy culture, it was vice versa. A slightly decrease in proteolysis with increasing heat impact was to be noted with both starter cultures. Concerning the relation of proteolysis to acidification, the fermentation process could be subdivided into three sections with different slopes if the non-ropy starter culture was used, while a linear relation was found if the ropy starter culture was applied. Regarding final product characteristics it was found that the functional properties of yoghurt decreased with increasing heat impact when the ropy starter culture was applied, while they remained to a far extent unchanged when the non-ropy starter culture was used. It can be concluded from these studies that a preheating of milk at a temperature of 85 degrees C (20 min) is optimal in regard to final yoghurt product characteristics.

Skim milk powders were prepared from control and transglutaminase-treated skimmed milk. The heat stability of reconstituted transglutaminase-treated skimmed milk (9.0% total solids) was markedly increased in the pH region of minimum stability (pH 6.8 to 7.1) compared with control milk, while the heat stability of reconstituted concentrated transglutaminase-treated skimmed milk (22.5% total solids) increased progressively as a function of pH relative to control milk. The effect of transglutaminase treatment on the heat stability of skimmed milk may have commercial applications, but extensive research is necessary to gain a better understanding of the mechanism by which transglutaminase improves heat stability.

Nucleotides, nucleosides and nucleobases belong to the non-protein-nitrogen (NPN) fraction of milk. The largest amounts of ribonucleosides and ribonucleotides--ribose forms only were considered in this review--were measured directly after parturition in bovine milk and other ruminants as well as in the milk of humans. Generally, concentrations of most of the nucleos(t)ides tend to decrease gradually with advancing lactation period or nursing time. The species-specific pattern of these minor constituents in milk from different mammals is a remarkable property and confirms, at least, the specific physiological impact of these minor compounds in early life. The physiological capacity of these compounds in milk is given by the total potentially available nucleosides. The main dietary sources of nucleos(t)ides are nucleoproteins and nucleic acids which are converted in the course of intestinal digestion into nucleosides and nucleobases the preferred forms for absorption in the intestine. Thus, nucleosides and nucleobases are suggested to be the acting components of dietary and/or supplemented nucleic acid-related compounds in the gut. They are used by the body as exogenous trophochemical sources and can be important for optimal metabolic functions. Up to 15 % of the total daily need for a breast-fed infant was calculated to come from this dietary source. Concerning their biological role they not only act as metabolites but are also involved as bioactive substances in the regulation of body functions. Dietary nucleotides affect immune modulation, e.g. they enhance antibody responses of infants as shown by a study with more than 300 full-term healthy infants. Dietary nucleos(t)ides are found to contribute to iron absorption in the gut and to influence desaturation and elongation rates in fatty acid synthesis, in particular long-chain polyunsaturated fatty acids in early stages of life. The in vitro modulation of cell proliferation and apoptosis has been described by ribonucleosides, in particular by modified components using human cell culture models. Due to the bio- and trophochemical properties of dietary nucleos(t)ides, the European Commission has allowed the use of supplementation with specific ribonucleotides in the manufacture of infant and follow-on formula. From the technochemical point of view, the ribonucleoside pattern is influenced by thermal treatment of milk. In addition ribonucleosides are useful indicators for quantifying adulterations of milk and milk products.

The in vitro modulation of apoptosis and cell proliferation by modified in comparison with non-modified ribonucleosides was investigated for the first time using peripheral blood lymphocytes, HL-60 cells and Caco-2 cells as human cell culture models. Modulating effects of several ribonucleosides were found in the range of 10(-7)-10(-3) mol/l. The following ribonucleosides induced significant apoptosis of HL-60 cells: adenosine, N6-dimethyladenosine, N6-(2-isopentenyl)-adenosine, N2-dimethylguanosine. A significant apoptotic effect on PBL was found with N6-dimethyladenosine and N6-(2-isopentenyl)-adenosine. N6-Dimethyladenosine, N6-(2-isopentenyl)-adenosine and guanosine had a pronounced inhibitory effect on Caco-2 cell apoptosis. Regarding the known function of ribonucleosides as pathobiochemical marker molecules for cancer, the possibility of a selective apoptotic effect against malignant cells is discussed.

Sodium caseinate is an effective substrate for transglutaminase. Crosslinking takes place at fast reaction rates resulting in high degrees of crosslinking. The polymers can be hydrolysed by trypsin, but the proteolysates contain residual portions of non- or partlyhydrolysed aggregates. Crosslinking of hydrolysed sodium caseinate decreased the number of free amino groups, but leads only to a very small increase in molecular weight of the peptides.

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of beta-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from alpha s-casein, were determined.

The aim of the study was to characterize the proteolytic properties of immobilized trypsin for obtaining phosphopeptide-rich fractions from casein. Trypsin was covalently bound to oxirane-acrylic beads. After incubation for 48 h immobilization degrees of about 85% were achieved. 20% of the immobilized enzyme exhibited no activity towards the substrate N-benzoyl-L-arginine ethyl ester. Compared with homogeneous catalysis with the soluble enzyme a 25% lower degree of proteolysis was calculated and a modified peptide pattern of the resulting proteolysates established. A caseinophosphopeptide (CPP) from alpha s1-CN (59-79) was not detectable after incubation with the carrier-bound enzyme. At a substrate concentration (S) of 15% (w/w) substrate saturation of the enzyme (E) was achieved. Increasing the substrate concentration to 20% (w/w) decreased the conversion rates (content of soluble amino-N) and the liberation of CPPs. Proteolysis of small (1% w/w) and partly also large (20% w/w) substrate concentrations (E/S = 1/100) is subject to changed kinetic conditions. The same was true for small and large enzyme concentrations (S = 5% w/w). Compared with enzyme saturation (E/S = 1/50), lack of enzyme (E/S = 1/800) led to a disproportional decrease in the proteolysis rate and to a markedly decreased content of hydrophobic peptides in the resulting proteolysates. Increasing the pH from 7.8 to 8.8 and the temperature from 37 degrees to 47 degrees C caused only a slight increase in conversion rates, but an overproportional liberation of CPPs (pH 8.8 = + 47%, 47 degrees C = +89%), in particular from beta-casein. Repeated use of immobilized trypsin resulted after nine runs in a loss in proteolytic activity and in CPP yields of approximately 25%, while the peptide pattern of the proteolysates remained qualitatively unchanged. Light microscopy shows that the oxirane-acrylic beads disintegrate to a large extent after nine repetitions.

Ribonucleosides are secreted as products of cellular RNA and ribonucleotide metabolism into physiological fluids such as blood, milk and urine. Unmodified and modified ribonucleosides have been detected in the micromolar range as minor constituents in the milk of different mammals. In addition to the common nucleosides adenosine, cytidine, guanosine, inosine and uridine, modified components such as 1-methyladenosine, 1-methylguanosine, 1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoyl-L-threonyladenosine, pseudouridine and 5-aminoimidazole-4-carboxamide-N-ribofuranoside (AICAR) have been identified and most of them quantified in samples of human and/or bovine and/or goat's milk. From these investigations it is known that nucleosides, in analogy to nucleotides, show a typical species-specific pattern. Longitudinal studies have been carried out to determine the concentration profiles of the individual ribonucleosides in the milk of humans as a function of the nursing time.

The primary function of dietary proteins is to supply the body adequately with indispensable amino acids and organic nitrogen. Little attention has been paid up to date to milk proteins, in particular caseins, that are currently the main source of biologically active peptides, although other animal as well as vegetable proteins are known to contain potentially bioactive sequences. Such regulatory peptides can be released by enzymatic proteolysis of caseins in vitro and in vivo and may act as potential physiological modulators of metabolism during the intestinal digestion of the diet. It has been proved that bioactive peptides derived from caseins, such as beta-casomorphins and phosphopeptides, can be released during gastrointestinal passage. It is also evident that peptides originating from food proteins should be taken into account as potential modulators of various regulatory processes in the body. The possible regulatory effects concern nutrient uptake (phosphopeptides, casomorphins), postprandial hormone secretion (casomorphins), immune defense (immunopeptides, casokinins, casomorphins) and neuroendocrine information transfer (casokinins). The advances in the research field of bioactive peptides are driven by a molecular understanding of biological processes, and analytical techniques are a critical component of this understanding. Different up-to-date methods, including peptide synthesis and immunochemistry, have been applied to the chemical characterization of bioactive peptides. Especially casein derived peptides have already found interesting applications, both as dietary supplements (phosphopeptides) and as pharmaceutical preparations (phosphopeptides, beta-casomorphins). The question of 'what kinds of bioactive peptides are beneficial and desirable as food constituents or as drugs' should be always carefully examined. However, the possibilities for the design of dietary products and 'natural' drugs look promising.

Phosphopeptides derived from casein may function as carriers for calcium and trace elements. In regard to such specific nutritive effects, the heat-induced changes in tryptic phosphopeptides liberated from bovine sodium caseinate as a model system were investigated. Both microwave and oven heating resulted in a marked loss of peptide-bound phosphorous (dephosphorylation) and a decrease of casein-phosphopeptides in the soluble part of the tryptic hydrolysate. It is concluded that hydrolysis of phosphoseryl to seryl residues was the prevailing degradation step to soluble proteolytic products, whereas lysinoalanyl-casein is claimed to be present almost exclusively in the pH 4.6-insoluble part of the tryptic digest.

Ribonucleosides are minor milk constituents and show a typical pattern which is assumed to be species-specific. As well as the unmodified components adenosine, cytidine, guanosine, inosine, and uridine, modified compounds such as Nl-methyladenosine and N6-carbamoylthreonyladenosine--products of the transfer RNA catabolism--have been identified and quantified in individual and bulk herd (race: German black pied) milk samples throughout a whole lactation period. The results of our longitudinal study have shown that--with the exception of the colostral phase--the levels of these minor constituents vary only slightly throughout lactation. These findings imply that ribonucleosides are useful for characterizing milk of different species and technological treatment. Ribonucleosides were determined and balanced, for example, in the course of the churning process, showing that the pattern of these minor milk constituents is useful as a "fingerprint" that allows differentiation between the three butter types defined in the German Federal Butter Ordinance.

The ribonucleosides adenosine, cytidine, guanosine, inosine and uridine as well as the modified components N1-methyladenosine and N6-carbamoylthreonyladenosine were characterized and determined quantitatively as minor constituents in raw bovine milk by use of an automated high performance liquid chromatography system. The studies have shown that except for the colostral phase the ribonucleoside levels are constant throughout the whole lactation period. That means, there is a typical ribonucleoside pattern which is assumed to be species-specific.

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.

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.

8-bromo-ATP is a substrate of nucleoside diphosphate kinase. Its reactivity is similar to that of ATP, and the reaction appears to be unaffected by the different conformations of these two substrates. The nucleoside diphosphate kinase reaction with 8-bromo-ATP cannot be monitored photometrically (optical test) using an NADH/NAD+-dependent enzyme system, because the product 8-bromo-ADP is not a substrate for pyruvate kinase. Measurements of the nucleoside diphosphate kinase reaction with 8-bromo-ATP therefore, were carried out by chromatographic analysis of the nucleoside-triphosphate/diphosphate ratio.

(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.

We propose a hypothetical model for the transmembrane exchange reaction catalysed by the mitochondrial adenine nucleotide carrier protein, which basically consists of an alternating reorientation of a transitory carrier-metal-nucleotide complex. The key features of the model are: the participation of an intrinsic divalent metal ion in the course of transport catalysis; the different stability constants of protonated and deprotonated nucleotide-metal complexes; the exposure and retraction of strategic arginyl residues; the alternating reorientation of the active center involving a change from the cytosolic conformation (Cc) to the matrix conformation (Cm).

Adenylate kinase activity of intact mitochondria is strongly inhibited by Ap5A, i.e. p1,p5-Di (adenosine-5') pentaphosphate, whereas oxidative phosphorylation is not affected. Therefore, Ap5A is a useful tool to distinguish between oxidative and non oxidative ATP generating reactions.

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.

Bioactive peptides have been identified as digestion products of several food proteins. All the bioactive sequences are hidden in an inactive state inside the polypeptide chain of the larger protein. Milk proteins are a rich source of biologically active peptides such as exorphins (casomorphins), phosphopeptides and immunopeptides. Such peptides are released during intestinal digestion of caseins and whey proteins. They may be involved in regulation of nutrient entry and influence the postprandial metabolism via stimulation of the secretion of hormones. Furthermore, they may exert a stimulating effect on the immune system. These findings offer new aspects for evaluating the nutritive value of food proteins. Moreover, bioactive peptides have already found interesting applications as dietary supplements and as pharmaceutical preparations.

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.

The ADP,ATP carrier of rat liver mitochondria is specifically inhibited by Meisenheimer-type trinitrophenyl (TNP) derivatives of ADP and ATP. Due to a systematic inhibition study we could show that the TNP-moiety itself, even in the 2', 3'-O-cyclic Meisenheimer complex, revealed no inhibition of mitochondrial ADP,ATP transport. Nucleosidic TNP-compounds are weak inhibitors. Introduction of a phosphate group at the 5'-position, however, enhances the inhibitory power markedly. Our findings point to a synergistic effect of the 5'-phosphate chain and the TNP-moiety. Irreversible inhibition by TNP-nucleotides can be ruled out to the fact that the inhibited ADP,ATP-transport system is fully reactivated by addition of albumin.

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.

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.

It was not possible to synthesize beta-nicotinamide-alpha-adenine dinucleotide (NalphaAD) via the NAD pyrophosphorylase-catalyzed reaction of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-triphosphate. It was therefore prepared by reacting a mixture of beta-nicotinamide mononucleotide and alpha-adenosine-5'-O-monophosphate in aqueous pyridine, using N,N'-dicyclohexylcarbodiimide as condensing agent. NalphaAD can replace NAD in the lactate-dehydrogenase-catalyzed oxidation of lactate to pyruvate with LDH 4M from hog muscle as well as LDH 4H from pig heart. Km values are found to be about one order of magnitude higher for NalphaAD than for NAD. The turnover number for NalphaAD with LDH 4H is similar to that of NAD (95%), whereas it is reduced to one third (35%) in the reaction with LDH 4M. Experiments findings are discussed on the lines of a NAD-equivalent conformation of NalphaAD in the holoenzyme complex.

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.

(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.

We describe an on-line multi-column high performance liquid chromatographic method for the selective clean-up and analysis of major and minor ribonucleosides in physiological fluids. Quantitative data obtained for the determination of some methylated ribonucleosides in human urines are compared with those obtained with the traditional off-line method. The on-line technique developed in our laboratory is distinguished from the off-line method by the following features: Sample clean-up and analysis of the target-compounds can easily be automatized, Total time of analysis, for example of urinary ribonucleosides, is decreased to 35 minutes, Laborious and error-prone evaporation and redissolution steps are avoided, Reliability of the overall analytical system can be controlled with ease, Small sample-volumes can be applied directly, Sensitive samples can be processed very rapidly under mild conditions, Results obtained with the on-line and off-line-techniques compare well.

We describe the group-selective separation and quantification of unmodified, modified and hypermodified ribonucleosides in physiological fluids (urine, serum) by on-line multidimensional high-performance affinity chromatography (HPAC)-reversed-phase liquid chromatography (RPLC). The excretion levels and patterns of ribonucleosides such as N1-methyladenosine, N1-methylinosine, N2-methylguanosine, N2-dimethylguanosine, N6-carbamoylthreonyladenosine and 2-pyridone-5-carboxamido-N-ribofuranoside were determined in urines from a control group and from patients with different diseases. The HPAC-RPLC method applied represents a powerful tool, e.g. as a non-invasive screening test, a method to investigate disorders in ribonucleoside and/or RNA metabolism, a method for drug monitoring during nucleoside chemotherapy, and a method to study renal ribonucleoside reutilization.

1. Solubility of mitochondrial membranes in various solvent systems was determined quantitatively. The most effective agent was the anionic detergent, sodium dodecylsulphate, which solubilizes 90% of the protein at the concentration of 0.1% followed by Triton X-100 (70%), sodium deoxycholate (60%), Brij 56 (50%), and guanidine hydrochloride (40%) at a concentration of 2 M. 2. Affinity chromatography of a clear 0.1% sodium dodecylsulphate solution of digitonized mitochondria on Sepharose 4B containing carboxyatractylate always resulted in the separation of two fractions, one of which was not retained by the column and the other which could be obtained after elution with 2% sodium dodecylsulphate. 3. The retained protein showed a high binding specificity for ATP and [3H]atractylate when compared with the unretained fraction. The amount of bound [3H]atractylate or carboxyatractylate-sensitive binding of ATP was 10.5 +/- 4 nmol/mg protein, and 22 +/- 8 nmol/mg protein, respectively. 4. The major component within the retained fraction, comprising 85% of the total weight, was protein, followed by phospholipids (14%) and approximately 1% triglycerides. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a major (95%) and a minor (5%) component with an apparent molecular weight of 26000 +/- 1000 and 8300 +/- 400, respectively. The gels did not stain for carbohydrates. Ultracentrifugal analysis showed a single, symmetrical boundry. 5. Double immunodiffusion analysis gave a single precipitin line with the corresponding antiserum. [14C]ADP exchange of digitonin particles was completely inhibited by an antiserum to the carboxyatractylate binding protein fraction, whereas the adenine nucleotide transport of intact mitochondria remained unaffected. In the presence of specific immunoglobulins state-3 respiration rate of digitonin particles was prolonged and reduced by approximately 25%. State-4 respiration rate was unaffected.

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 describe an on-line multi-dimensional column chromatography system. It consists of a high-performance liquid affinity chromatography perfractionation column for cis-diol compounds and a series-connected reversed-phase high-performance liquid chromatography system for the analysis of ribonucleosides. This on-line procedure allows the rapid and direct analysis of methylated ribonucleic acid catabolites in biological fluids (serum, urine) and therefore might be useful in pathobiochemistry.

We describe the selective analysis of N-methylated ribonucleosides, i.e., N1-methyl adenosine (m1Ado) and N7-methyl guanosine (m7Guo) in the presence of other nucleosides in urine. m1Ado as well as m7Guo were characterized by use of chromatographic techniques including substance specific chemical reactions.