Coupled Enzyme System Research Articles

Subcellular compartmentation in the synthesis of the milk sugars lactose and ?-2,3-sialyllactose

This paper assembles published and new material to trace out the compartmental aspects of milk sugar synthesis in the mammary gland. The generation of lactose, from glucose and UDP-galactose, in thetrans cisternae of the Golgi stack is well established from biochemical and electron microscopy work. New experiments show that in the rat essentially all this lactose is available for sialyllation to α-2,3-sialyllactose, which accumulates in the same compartment. UDP arising from utilization of UDP-galactose inhibits lactose synthetase, but is removed by nucleoside diphosphatase. Therefore lactose synthetase, sialyltransferase and nucleoside diphosphatase form a coupled enzyme system located on the luminal face of the Golgi membrane. Transport of UMP to the cytosol, followed by its re-conversion via UDP and UDP-glucose to UDP-galactose, defines the operation of a uridine nucleotide cycle that links Golgi and cytosol compartments in the support of milk sugar synthesis. Evidence for carrier-mediated uniport or antiport of UMP and UDP-galactose across the Golgi membrane is discussed, together with the evidence for transport of glucose and inositol by small water-filled pores that forbid back diffusion of lactose, galactinol or sialyllactose. Possible intra-Golgi signals, achieving short-term regulation of milk sugar synthesis, include glucose, submicromolar free Mn(II) ions and cationic activator proteins. Experimental evidence for intracellular and intra-Golgi glucose concentrations in the range 0.1–0.4 mM imply lack of saturation of lactose synthetase. It is shown that lactose synthetase within undamaged Golgi membrane vesicles is about 100 times more sensitive to Mn ions than in damaged or solubilized membranes. Both lactose synthetase and sialyltransferase are greatly stimulated by basic proteins, and similar lactose synthease-activating material is extractable from Golgi preparations. Development of these features may designate the sorts of control exerted upon these Golgi enzymes, and perhaps others. This may uncover the mechanisms by which hormonal and nutritional changes, and manipulation of mammary tissue in vitro, cause profound alterations in milk sugar production.

Biochemical Parameters of Initiation and Regulation of Sperm Motility

Studies of in vitro models demonstrate that a forward motility protein (FMP) is required for the initiation of forward motility in the immature epididymal spermatozoa. FMP is a heat-stable glycoprotein derived from epididymal plasma. During the epididymal maturation of spermatozoa in vivo, there is a marked increase of intrasperm pH and level of cyclic adenosine monophosphate (cAMP). Several studies suggest that exogenous FMP in concert with elevated intrasperm pH and level of cAMP initiates flagellar motility during the epididymal transit of sperm. cAMP activates sperm cytosolic cAMP-dependent protein kinases, which in turn phosphorylate multiple intrasperm phosphoproteins that may regulate flagellar motility. Exogenous calcium ion activates intact sperm motility, although it inhibits motility of demembranated cells on reactivation. Occurrence of cAMP-dependent type I and II protein kinases, a novel cAMP-independent protein kinase, and a phosphoprotein phosphatase has been demonstrated on the external surface of spermatozoa. The sperm surface has a coupled-enzyme system: ecto-cAMP-independent protein kinase and phosphoprotein phosphatase that regulate the phosphorylation and dephosphorylation of endogenous sperm ectophosphoproteins. The specific activities of these ecto-enzymes increase markedly during forward progression, suggesting that they may have a role in regulating flagellar motility.

A Stochastic Model for Coupled Enzyme System

AbstractThe molecular biology of transformed cancer cells singles out key enzymes as sensitive targets of anti‐cancer drugs. Here we use one substrate–one intermediate–one final product model for a coupled enzyme system. The transfer rates for the mechanism are taken as continuous but subject to random fluctuations. Explicit formulae for the first moments of the distribution of the process are obtained. These formulae allow us to take into account not only the variability between the subjects, but also the variability of the process for a single subject. The present results allow us also to build the prediction interval for a particular time period given the observations for some preceding moments.

31P magnetization transfer in the phosphoglyceromutase-enolase coupled enzyme system.

The rate of exchange of phosphoryl groups between 2-phosphoglycerate, 3-phosphoglycerate and phosphoenolpyruvate by the coupled phosphoglyceromutase-enolase enzyme system using one- and two-dimensional 31P NMR spectroscopy was measured. Magnetization exchange in one-dimensional experiments was achieved by saturation transfer with selective irradiation at both one and two sites in this three-site exchange system using the DANTE pulse sequence. The two-dimensional magnetization exchange experiment avoids the need to selectively saturate at one or more frequencies which may be difficult in complex exchange systems. Analysis of the two-dimensional exchange experiment by the back transformation method yielded exchange rate constants in good agreement with the saturation transfer method.

A general coupled spectrophotometric assay for decarboxylases

A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO 2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to l-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase. The method is capable of detecting the liberation of as little as 1 nmol of CO 2/min and was tested in assays of lysine decarboxylase, orotidine-5′-phosphate decarboxylase, and 4′-phosphopantothenoyl- l-cysteine decarboxylase.

A fluorescence assay for α-1,2-mannosidases involved in glycoprotein processing reactions

A highly specific, sensitive, and conveient fluorescence assay for α-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O-methyl-2- O-α- d-mannopyranosyl-α- d-mannopyranoside by the α-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated α-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the α-1,2 linkage. The mannosyl linkages in α-1,3- and α-1,6-linked methyl-disacchardes, in methyl-α- d-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the α-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The K m for the α-1,2-linked methyl-disaccharide is 0.5 m m.

Multiple Steady States in a Coupled Enzyme System Represented by the Enzymatically Catalyzed Production of l‐Phenylalanine

Multiple Steady States in a Coupled Enzyme System Represented by the Enzymatically Catalyzed Production of <scp>l</scp>‐Phenylalanine

Depyrophosphorylation of adenosine 5′-triphosphate (ATP)

The slow hydrolysis of free adenosine 5′-triphosphate (ATP) in both weakly and strongly basic aqueous solution (NaOH, 4 °C) is found to favor depyrophosphorylation over dephosphorylation. In the middle pH region (25 °C) the addition of N 4Co(H 2O) 2 3+ (N 4=tn 2, trpn; N 4Co(H 2O)(OH) 2+ pre-dominant species) to preformed N 4CoATP − complexes (N 4 = tn 2, trpn, tren) results in increased rates in production of P i, but not of PP i. However, addition of Cu 2+ or Ca 2+ to preformed N 4CoATP − complexes (N 4 = tn 2, trpn, tren) leads to increased rates in the production of PP i as well as of P i. Mechanistic implications are discussed. The production of P i from ATP (dephosphorylation) has been monitored by quenching aliquots with Eu 2+ (H +) and rapidly replacing multivalent cations with Na + (ion exchange) before developing phosphomolybdate for analysis. The production of PP i from ATP (depyrophosphorylation ) has been monitored, following similar quenching with Eu 2+ (H +) and replacement of multivalent cations, by assaying for free PP i using a coupled enzyme system.

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [γ- 32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.

43] Preparation of stereospecifically labeled porphobilinogens

Publisher Summary This chapter deals first with the use of labeled succinic acid derivatives, which introduce label into the acetate and propionate side chains of porphobilinogen (PBG), and then with the use of labeled glycine, which is incorporated into the aminomethyl side chain of PBG. The prototype methods are also described and possible variations are considered briefly. Labeling of the acid side chains is achieved biosynthetically with the stereospecific label originating from succinic acid. The labeled succinic acid is prepared in turn by decarboxylation of a stereospecifically labeled 2-oxoglutarate. As these syntheses pass through a succinic acid intermediate, symmetrical apart from the isotopic labeling, the final labeling is distributed equally between C-2 and C-3 of the aminolevulinic acid (ALA), and hence between C-6, C-8, and C-9 of the PBG. The label from [2-3H2]glycine is stereospecifically incorporated into C-5 of ALA. The stereospecifically labeled ALA thus formed may be trapped to give PBG stereospecifically labeled in the aminomethyl sidechain. The preparative procedure is similar to the coupled enzyme system described in the chapter, except that incubation conditions are chosen to maximize the radiochemical incorporation of glycine rather than succinyl CoA, by reducing the glycine concentration from about 130 to 3 mM (Km about 12 mM), and the reaction is performed in phosphate buffer rather than the Tris buffer. The complications arising from the nonenzymatic racemisation of the chiral center at C-5 of ALA catalyzed by pyridoxal phosphate (required as cofactor by the ALA synthetase) are avoided by using as low a concentration of pyridoxal phosphate as possible.

A kinetic study of the irreversible inhibition of an enzyme measured in the presence of coupled enzymes. Fluorescein isothiocyanate as inhibitor of the adenosinetriphosphatase activity from sarcoplasmic reticulum

A systematic procedure for the kinetic study of irreversible inhibition, when the enzymatic activity is measured in the presence of a coupled enzyme system, has been developed and analyzed. Simultaneous variation of the enzyme and inhibitor concentrations, maintaining a constant ratio between them, is recommended. The methodology is established to estimate the kinetic constants corresponding to the irreversible inhibitor. This approach is illustrated by the study of the inhibition of fluorescein isothiocyanate on the Ca 2+-ATPase activity from sarcoplasmic reticulum measured in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. Treatment of the experimental data has been carried out by non-linear regression.

Inhibition of Mucopolysaccharide Sulfation Using Rabbit Liver Sulfotransferases

A method of measuring the inhibition of sulfation of mucopolysaccharides employed the coupled-enzyme system of Wortman. The procedure utilized the phenol and mucopolysaccharide sulfotransferases of rabbit liver extract. Compounds tested as inhibitors included substituted salicyclic acids and related compounds, some hydroxamic acids, other agents capable of metal-ion complexation, and miscellaneous compounds, mostly containing sulfur. The most effective inhibitors were vanillin oxime, salicylhydroxamic acid, and other substituted salicylic acid derivatives of weaker acid strength than salicylic acid.

Enzymatic synthesis of trehalose from maltose.

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 μg. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 μg. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.

Assay of D-Fructose 2,6-Bisphosphate in Biological Extracts

Abstract Tissues or cells are extracted in mildly alkaline solutions and reduced by sodium-borohydride. The fructose 6-phosphate released from the intact D-fructose 2,6-bisphosphate by gentle acid hydrolysis is determined fluorimetrically in a coupled enzyme system with phosphoglucoisomerase (EC 5.3.1.9), NAD+: glucose 6-phosphate dehydrogenase (EC 1.1.1.49), diaphorase (EC 1.6.99.1) and resazurine. To demonstrate the application of this method, the concentration of D-fructose 2,6-bisphosphate in liver, yeast and legume-seedlings extracts at different physiological states has been measured and compared to the existing pool level of other intermediates of glycolysis.

Mechanism and stereochemistry of the porphobilinogen deaminase and protoporphyrinogen IX oxidase reactions: stereospecific manipulation of hydrogen atoms at the four methylene bridges during the biosynthesis of haem.

[2,11S-3H2;2,11-14C2] Porphobilinogen (PBG) with an enantiomeric excess greater than 81 % was prepared in a coupled enzyme system from [2RS-3H2;2-14C2]glycine. Incorporation of this chiral PBG into haem using a broken cell enzyme system from chicken blood showed retention of only one tritium per haem molecule. Degradation of the haem showed that tritium had been incorporated specifically into the C-10 position of haem. Control experiments with [11RS-3H2;2,11-14C2]-PBG showed an equal distribution of tritium at all four meso positions of haem. The implications of this result are discussed in terms of the mechanism of two enzymes in haem biosynthesis: porphobilinogen deaminase (hydroxymethylbilane synthase) and protoporphyrinogen IX oxidase.

22] Microassay procedure for NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

This chapter presents the microassay procedure for nicotinamide adenine dinucleotide (NAD) + -dependent 15-hydroxyprostaglandin dehydrogenase. The assay procedure is based upon the determination of the amount of substrate prostaglandin (PG) E 1 lost during a reaction with NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (NAD + - PGDH) in the tissue sample. The reaction is carried out in a small reaction volume at 37° for the specified time, then terminated. The amount of unreacted PGE 1 is measured in a coupled enzyme system. The initial reaction may be carried out on nanogram quantities of freeze-dried tissue in a reaction volume of 0.1 μL suspended in oil. Conversely, the microassay may be applied to larger sample sizes in a 1-ml reaction volume. The quantitative histochemical assay consists of seven steps. The procedure is described for NAD + -PGDH in kidney.

Enzymatic Determination of Triglycerides in Conjunction with High Performance Liquid Chromatography

Abstract The glycerol kinase (GK) catalyzed reaction involving the conversion of glycerol and adenosine triphosphate (ATP) to glycerol-3-phosphate and adenosine diphosphate (ADP) has been used in conjunction with HPLC for the determination of triglycerides. After alkaline hydrolysis of the triglycerides to glycerol, the enzyme reaction was carried out. The ADP formed and the remaining ATP were then separated by HPLC and the ADP peak area correlated to the concentration of triglycerides originally present in the sample. Linearity of the method was established from 28–180 mg/dl with a reproducibility of 6.5% RSD. A comparison between the HPLC method and the standard coupled enzyme system for triglycerides in real serum indicated a correlation coefficient of 0.977.

An isoenergetic exchange mechanism which accounts for tubulin-GDP stabilization of microtubules.

We have developed a coupled enzyme system composed of hexokinase, glucose, and nucleoside diphosphate kinase which is able to rapidly convert GTP at the exchangeable nucleotide binding site of tubulin to GDP. Using this method, we have studied the dynamic properties of microtubules and tubulin subunits in the presence of GDP. Conversion of GTP to GDP causes microtubules to change to a new, somewhat lower steady state level; dilution studies show that subunits disassemble from microtubules at steady state in the presence of GDP at a rate comparable to that in the presence of GTP; in reactions of existing microtubules with tubulin-GDP subunits it was found that tubulin-GDP subunits do not participate in net microtubule elongation. From these observations we conclude that in the presence of tubulin-GDP the microtubule steady state has an unusual property; in spite of the fact that the microtubule is continuously undergoing rapid subunit loss, but not subunit addition, a nearly constant steady state level of microtubule mass is maintained. This must mean that tubulin-GDP subunits, although unable to participate in a net addition, can participate in additions each of which compensates for the dissociation of a single subunit from the microtubule. This is equivalent to the existence of an isoenergetic exchange of a tubulin-GDP in solution for a subunit which had been lost from the end of a microtubule.

An improved rapid assay for S-adenosyl-L-homocysteine hydrolase.

A coupled enzyme system was devised to assay S-adenosyl-L-homocysteine hydrolase activity spectrophotometrically and to stain the hydrolase selectively in polyacrylamide gels. The assay procedure monitors the formation of uric acid derived from the catabolism of the adenosine moiety of S-adenosylhomocysteine. The staining procedure allows the determination of the molecular weight of the hydrolase when extracts are electrophoresed on polyacrylamide gradient gels and compared to standard of known molecular weight. The specificity of the enzyme for the homocysteine moiety was also investigated by testing modified compounds as substrates. All the analogs tested were inactive as substrates for hydrolysis, indicating a strict specificity.