David Shugar

The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme

Abstract 1. 1. A rapid and reproducible method for measurement of lysozyme activity is described. 2. 2. Using this method, the ultra-violet inactivation of lysozyme has been studied and found to conform to a first order reaction. 3. 3. The measured quantum yield for inactivation at 2537 A is 0.024 and remains unaltered over the pH range 3.6 to 12.0. A small effect of concentration on quantum yield is indicated. 4. 4. Inactivation is accompanied by photo-oxidation and other secondary processes, with no apparent liberation of free amino acids or peptides. 5. 5. The relation of molecular weight to quantum yield, as well as the other findings, are discussed.

Purine nucleoside phosphorylases: properties, functions, and clinical aspects

The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. This review updates the properties of the enzymes from eukaryotes and a wide range of prokaryotes, including a tentative classification of the enzymes from various sources, based on three-dimensional structures in the solid state, subunit composition, amino acid sequences, and substrate specificities. Attention is drawn to the compelling need of quantitative experimental data on subunit composition in solution, binding constants, and stoichiometry of binding; order of ligand binding and release; and its possible relevance to the complex kinetics exhibited with some substrates. Mutations responsible for PNP deficiency are described, as well as clinical methods, including gene therapy, for corrections of this usually fatal disease. Substrate discrimination between enzymes from different sources is also being profited from for development of tumour-directed gene therapy. Detailed accounts are presented of design of potent inhibitors, largely nucleosides and acyclonucleosides, their phosphates and phosphonates, particularly of the human erythrocyte enzyme, some with Ki values in nanomolar and picomolar range, intended for induction of the immunodeficient state for clinical applications, such as prevention of host-versus-graft response in organ transplantations. Methods of assay of PNP activity are reviewed. Also described are applications of PNP from various sources as tools for the enzymatic synthesis of otherwise inaccessible therapeutic nucleoside analogues, as coupling enzymes for assays of orthophosphate in biological systems in the micromolar and submicromolar ranges, and for coupled assays of other enzyme systems.

Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 ('casein kinase-2').

The specificity of 4,5,6,7‐tetrabromo‐2‐azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase‐2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr‐ or Tyr‐specific. In the presence of 10 μM TBB (and 100 μM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3β and cyclin‐dependent kinase 2/cyclin A) underwent moderate inhibition, with IC50 values one–two orders of magnitude higher than CK2 (IC50=0.9 μM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell‐based assays.

Conformation of the exocyclic 5′-CH2OH in nucleosides and nucleotides in aqueous solution from specific assignments of the H5′, and H5″ signals in the NMR spectra

Abstract Analyses of published NMR spectra for uridine, β-pseudouridine and their 3′-monophosphates, together with the deshielding effect of a phosphate group, have made possible specific assignments of the signals of the two 5′ protons. This permits of a more accurate evaluation of the relative populations of the three CH 2 OH conformers and, in turn, of a suitable choice of the constants in the Karplus relation applicable to this type of system. The results have been extended to a variety of other nucleosides and nucleotides, and to a consideration of the nature of the deshielding effect of a phosphate group on the ribose protons.

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.

Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine

The properties of two unusual substrates of calf spleen purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1), 7-methylguanosine and 7-methylinosine, are described. The corresponding bases, 7-methylguanine and 7-methylhypoxanthine, are neither substrates in the reverse, synthetic reaction, nor inhibitors of the phosphorolysis reaction. Both nucleosides exhibit fluorescence, which disappears on cleavage of the glycosidic bond, providing a new convenient procedure for continuous fluorimetric assay of enzymatic activity. For 7-methylguanosine at neutral pH and 25 degrees C, Vmax = 3.3 mumol/min per unit enzyme and Km = 14.7 microM, so that Vmax/Km = 22 X 10(-2)/min per unit as compared to 8 X 10(-2) for the commonly used substrate inosine. The permissible initial substrate concentration range is 5-100 microM. Enzyme activity may also be monitored spectrophotometrically. For 7-methylinosine, Vmax/Km is much lower, 2.4 X 10(-2), but its 10-fold higher fluorescence partially compensates for this, and permits the use of initial substrate concentrations in the range 1-500 microM. At neutral pH both substrates are mixtures of cationic and zwitterionic forms. Measurements of pH-dependence of kinetic constants indicated that the cationic forms are the preferred substrates, whereas the monoanion of inosine appears to be almost as good a substrate as the neutral form. With 7-methylguanosine as substrate, and monitoring of activity fluorimetrically and spectrophotometrically, inhibition constants were measured for several known inhibitors, and the results compared with those obtained with inosine as substrate, and with results reported for the enzyme from other sources.

Selectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.

Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

Abstract It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD+ (or NADP+). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H2O2 at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD+ is about 75%. Under analogous irradiation conditions, the dimers (NAD)2 and (NADP)2 undergo disproportionation to NAD+ and NADP+, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)2.

The structure of poly-5-methylcytidylic acid and its twin-stranded complex with poly-inosinic acid

An examination of the temperature profiles of both the neutral and acid forms of poly-5MeC † indicates that they possess properties analogous to those of the corresponding forms of poly-C. In particular, the neutral forms of both poly-C and poly-5MeC exhibit appreciable hyper chromicity to the red of 280 mμ, which is increased at elevated temperatures; the importance of this phenomenon is discussed in relation to theories of the origin of hyperchromicity. The twin-stranded poly-(I + 5MeC) is considerably more stable than poly-(I + C), the former possessing a T m about 18°C higher than the latter. This is strikingly similar to the 20°C difference in T m between poly-(A + 5MeU) and poly-(A + U), so that the influence of a 5-methyl substituent in the pyrirnidine rings is approximately the same in both helices. The stabilizing effect of a 5-methyl substituent is discussed in detail in relation to other synthetic polynucleotide analogues and with respect to natural nucleic acids, including those containing base analogues other than those normally encountered.

Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (pKa = 8.05, phi= 0.55), as is also the case for 8-azaIno (pKa = 8.0, phi = 0.02), whereas 8-azaAdo is a strong emitter (phi = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG (phi = 0.05 to 0.33, dependent on lambda exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10-15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial (Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, alpha-D-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorolysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno (Ki approximately 20 microM), also the only one to inhibit phosphorolysis by the calf spleen enzyme (Ki approximately 40 microM). The nature of this inhibition is apparently uncompetitive.