The enzymatic measurement of adenine nucleotides and P-creatine in picomole amounts

Enzymatic methods are described for the analysis of ATP, ATP + ADP, total adenylates, or P-creatine in biological samples. The methods include (i) direct fluorometric procedures for the measurement of 0.1–10 nmol using hexokinase and glucose-6-P-dehydrogenase as the indicator step; (ii) an enzymatic cycling procedure with a sensitivity of 1–50 pmol; and (iii) the measurement of light emission in the luciferin-luciferase system with a sensitivity of 0.1–80 pmol.     

N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

The effects of adenine nucleotides on pyruvate metabolism in rat liver

1. The effects of adenine nucleotides on pyruvate metabolism by isolated liver cells and isolated mitochondria have been investigated. The amount of pyruvate carboxylated has been estimated by determining the tricarboxylic acid-cycle intermediates, glutamate and aspartate accumulating in the incubation medium. The extent of pyruvate oxidation has been assessed by measuring oxygen uptake and the yield of ¹⁴CO₂ from [1-¹⁴C]pyruvate and [2-¹⁴C]pyruvate. 2. When catalytic amounts of adenine nucleotides (1–2mm) were added to suspensions of isolated liver cells incubated with pyruvate an ATP:ADP ratio greater than 6:1 was maintained. Both pyruvate oxidation to acetyl-CoA and the oxidation of acetyl-CoA through the tricarboxylic acid cycle were stimulated but pyruvate carboxylation was not affected. The production of acetyl-CoA exceeded the capacity of the cells for the oxidation of acetyl-CoA and the excess was converted into ketone bodies. 3. If a low ATP:ADP ratio was maintained in isolated cells or mitochondria by incubating them with dinitrophenol or hexokinase, pyruvate carboxylation was grossly inhibited, oxygen uptake depressed and ketone-body formation stimulated. Measurement of oxaloacetate concentrations confirmed that under these conditions oxaloacetate was rate-limiting for the oxidation of acetyl-CoA via the tricarboxylic acid cycle. The inclusion in the incubation medium of fumarate (1·25mm) completely prevented the ketogenic action of dinitrophenol or hexokinase. 4. When ADP (5mm) was added to a suspension of isolated liver cells incubated with pyruvate an actual ADP concentration of about 1mm was attained. This brought about effects on pyruvate metabolism similar to those obtained with dinitrophenol or hexokinase. 5. These results support the concept that the relative concentrations of adenine nucleotides within the liver cell may play a role in governing the rates of pyruvate oxidation and carboxylation. In addition, they provide further evidence that the availability of oxaloacetate in the liver cell can play a key role in determining whether acetyl-CoA arising from pyruvate is oxidized through the tricarboxylic acid cycle or converted into ketone bodies.     

Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells.

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.     

Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate.

Polypeptides coupled with dimethylaminoazobenzene isothiocyanate through their amino groups to form dimethylaminoazobenzenethiocarbamoyl- (DABTC-)peptides can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). As little as 1 ng (2 pmol) of a DABTC-pentapeptide can be identified against a stable base-line with the signal-to-noise ratio of 10. The DABTC-peptides can also be recovered from the column, and their N-terminal amino acids (obtained by direct treatment with aqueous acid) and amino acid compositions and sequences can be all analysed at the picomole level. The power of this method is demonstrated by the complete separation and characterization of model peptides, peptide hormones and peptides derived from enzymic fragmentation of proteins. This new technique should provide a sensitive and efficient tool for peptide analysis at the nanogram level.     

Analysis of NAD+ in picomole amounts with sodium [32P]pyrophosphate and NAD-pyrophosphorylase.

A new method is described for the determination of NAD⁺ in picomole amounts. An enzymatic coupling system of NAD-pyrophosphorylase and hexokinase is used to convert sodium [³²P]pyrophosphate and NAD⁺ to [³²P]ADP, glucose 6-[³²P]phosphate, and NMN. The key step in this analysis is the selective adsorption of the reaction product [³²P]ADP, onto activated charcoal with a solution of 1m K₂HPO₄:10% trichloroacetic acid (1:3, v/v, pH 2). The range of concentrations of NAD⁺ that can be measured is 1–200 pmol. The simplicity of the method allows as many as 180 samples to be assayed in 4–5 h. This procedure has been used to quantitate NAD⁺ in crude extracts of germinating wheat embryos.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Reactions of dysprosium with adenine nucleotides

Dysprosium catalyzes a rapid hydrolysis of both ATP and ADP, at ambient temperatures, pH 7.0, where no hydroxide precipitates. The reactive complexes, at pH 6.7, were found to contain 2Dy:1ATP and 3Dy:2ADP. AMP forms an insoluble complex containing 1Dy:2AMP, which does not hydrolyze. ATP also forms a soluble 1Dy:1ATP complex, which does not react. Dysprosium only catalyzes the hydrolysis of ATP above pH 5.8, where it has been titrated to the hydroxide. At the optimum pH (pH 7) the stoichiometric composition is Dy2.ATP.(OH)2, indicating that the active complex is neutral, whereas at pH 5.8 the stoichiometric composition is Dy2.ATP.(OH)+, indicating an inactive cationic complex. The mechanism proposed for the hydrolysis is consistent with those proposed for other in vitro systems known to catalyze the hydrolysis of ATP.     

Protein quantitation at the picomole level: an O-phthaldialdehyde-preTSK column-derivatization assay

A fluorescent protein assay was described wherein an isocratic high-performance liquid chromatography system was used to separate the o-phthaldialdehyde-derivatized proteins from interfering components. Using a small TSK guard column equilibrated in 0.1% sodium dodecyl sulfate, it was demonstrated that all proteins and peptides examined, containing more than 22 residues, coelute in the excluded volume and were resolved from fluorescent signals contributed by commonly used reagents. The assay was linear over a useful range of 3 ng to 1 microgram of protein and required less than 15 microliter of sample.     

Method for the detection of glycopeptides at the picomole level in HPLC peptide maps.

Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.     

Quantitative amino acid compositions at the picomole level using fluorescence scanning

Simple techniques are described for quantitating dansyl amino acids by direct fluoresence scanning and by photo-copying and densitometry. This technique is accurate between 1.0 to 10 × 10^?12 moles with a lower limit of detection around 1 × 10^?14 moles. It is possible to obtain amino acid compositions on very small quantities of peptides and it seems likely that this method will be useful for automatic peptide sequenators which use subtractive dansyl-Edman degradation as a detection procedure.     

Annexin VI interacts with adenine nucleotides and their analogs.

Annexin VI (AnxVI), a member of the annexin family of Ca2+- and membrane-binding proteins, has been shown to interact in vitro with adenine nucleotides. Furthermore, it has been proposed that within the AnxVI molecule a nucleotidde-binding domain exists, which is located in the C-terminal half of the protein, in the vicinity of Trp343. By comparison of exposure of tryptophan and multiple tyrosine residues upon nucleotide binding, as revealed by quenching of intrinsic fluorescence of AnxVI by ATP, ADP or cAMP, it can be concluded that the binding of nucleotides evokes changes in the protein tertiary structure. Moreover, in the course of present study we have found that AnxVI binds to a non-hydrolysable analog of ATP, the triazine dye Cibacron blue 3GA (CB3GA), immobilized on agarose. Binding reveals negative cooperativity with respect to protein concentration and is Ca2+-dependent. Binding is prevented by ATP. CB3GA binds to AnxVI also in solution, evoking the formation of annexin multimers. On the basis of this observation it can be suggested that interaction of CB3GA with AnxVI is useful to examine, with some limitations, the self-association of annexin molecules implying to play a role in interacting of AnxVI with biological membranes.     

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.