d(-)-Lactate dehydrogenase from Allomyces

Summary d(-)-lactate dehydrogenase from hybrid male strain of Allomyces has been partially purified.The enzyme shows multiple binding sites for NADH. It obeys Michaelis-Menten kinetics for pyruvate. The inhibition of the enzyme activity by ATP is of mixed type. ADP is not an allosteric inhibitor of the enzyme. AMP and cyclic 3,5-AMP do not affect the enzyme. NAD⁺ acts as a product inhibitor.     

Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae

Summary Glycogen phosphorylase of pupal fat body of the silkmoth,Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylasea than assay by release of P_i from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylasea equally. TheK _ms for P_i were estimated as 5 mM for phosphorylasea in the absence of AMP and 18 mM for phosphorylaseb with 2 mM AMP.When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to thea form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling.Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as shock activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C (cold activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced.In fat body homogenates, phosphorylaseb was converted to phosphorylasea by incubation with ATP and Mg²⁺, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase.Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels.It is concluded that the conversion of silkmoth pupal fat body phosphorylaseb to phosphorylasea can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.Abbreviations DTT dithiothreitol - cyclic AMP 3,5-cyclic adenosine monophosphate - cyclic GMP 3,5-cyclic guanosine monophosphate - P _i inorganic phosphate This investigation was begun in the Department of Biology, Yale University, New Haven, Connecticut, USA     

Histochemical studies on phosphorylase activity in the tissues of the albino rat under normal and experimental conditions

Summary Various substrate mixtures have been tested for the histochemical demonstration of phosphorylase in tissue blocks. These studies indicate that phosphorylase activity, cytological detail and localization of the reaction product are optimally demonstrated when tissue blocks are incubated for one hour or longer in a medium containing high concentrations of G-1-P, ATP and PVP.Abbreviations AMP adenosine-5-monophosphate - ATP adenosine-5-triphosphate - EDTA ethylenediamine-tetraacetic acid - G-1-P glucose-1-phosphate - PPa active phosphorylase - PPb inactive phosphorylase - PVP polyvinyl pyrrolidone     

Re-evaluation of the specificity of adenylyl (beta,gamma-methylene)diphosphonate as a substrate for adenylate cyclase.

Summary The conversion of the ATP-analogue adenylyl(,-methylene)diphosphonate (AMPPCP) to cyclic AMP by adenylate cyclase of rat liver membranes was demonstrated using a radioimmunoassay for cyclic AMP. The conversion was only insignificantly lower than with adenylylimidodiphosphate (AMPPNP), another ATP-analogue which is usually used in the histochemical adenylate cyclase assay. The unspecific phosphate production was lower with AMPPCP as compared to AMPPNP. Therefore AMPPCP is considered to be a more suitable substrate for the histochemical assay.Unspecific phosphate deposition in the histochemical assay was due to ATP:pyrophosphatase activity and could be significantly inhibited by 1mm NAD. However, a residual phosphate deposition due to cleavage of NAD could not be suppressed. Adenylate cyclase activity could be markedly activated by 5×10^–5 m forskolin, an activator of the catalytic subunit of the enzyme, and inhibited by 1mm 25-dideoxyadenosine, a specific inhibitor of adenylate cyclase. Adenylate cyclase was localized predominantly in the sinusoidal part of the plasma membrane, while ATP-pyrophosphatase seemed to be restricted to the canalicular part. It is concluded that at least three parallel assays are necessary for routine histochemical demonstration of adenylate cyclase, namely (1) basal activity (2) activation by forskolin and (3) inhibition by 25-dideoxyadenosine, to demonstrate a specific enzyme reaction.     

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes

Summary The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E₁ both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha₁-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha, and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.Abbreviations cAMP Adenosine-3,5-Cyclic Monophosphate - cGMP Guanosine-3,5-Cyclic Monophosphate - G_i, G_S Guanine nucleotide-binding proteins linked to inhibition and stimulation, respectively, of adenylate cyclase - GTP Guanosine-5-triphosphate - PDE Cyclic Nucleotide Phosphodiesterase - PGE₁ Prostaglandin E₁     

Acetylcholinesterase molecular forms from rat and human erythrocyte membrane

Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N⁶, O²-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PK_I) decreased relative to cyclic AMP-dependent protein kinase type II (PK_II) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PK_I, PK_II and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP N⁶,O²-dibutyryladenosine-3, 5-cyclic monophosphate - cyclic AMP adenosine 3,5-cyclic monophosphate - PK_I type I cyclic AMP-dependent protein kinase - PK_II type II cyclic AMP-dependent protein kinase     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Stereoselective aminoacylation of a dinucleoside monophosphate by the imidazolides ofDl-alanine and N-(tert-butoxycarbonyl)-Dl-alanine

Summary The aminoacylation of diinosine monophosphate (IpI) was studied. When the acylating agent was the imidazolide of N-(tert-butoxycarbonyl)-Dl-alanine, a 40% enantiomeric excess of thel isomer was incorporated at the internal 2 site and the positions of equilibrium for the 23 migration reaction differed for theD andl enantiomers. The reactivity of the nucleoside hydroxyl groups decreased in the order 2(3)>internal 2>5, and the extent of reaction was affected by the concentration of the imidazole buffer (pH 7.1). In contrast, reaction of IpI with the imidazolide of unprotectedDl-alanine led to an excess of theD isomer at the internal 2 site, while reaction with the N-carboxy anhydride ofDl-alanine proceeded without detectable stereoselection. The relevance of these results to the evolution of optical activity and the origin of genetically directed protein synthesis is discussed.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Oligomerization of 3′-amino-3′-deoxyguanosine-5′-phosphorimidazolidate on a d(CpCpCpCpC) template

Summary 3-Amino-3-deoxyguanosine-5-phosphorimidazolidate (ImpGnh ₂) oligomerizes more rapidly and regiospecifically than related nucleotide derivatives on a d(CpCpCpCpC) template. The greater nucleophilicity of the amino group leads to efficient oligomerization even when the structure of the double-helical complex formed by the template and the substrate is not optimal for reaction. The use of amine-containing analogues should permit us to develop models of potentially prebiotic polymerization reactions that cannot be studied easily using natural nucleotides.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Kinetics of DIDS inhibition of swelling-activated K-Cl contrasport in low K sheep erythrocytes

Summary The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocy-anatostilbenes H₂DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC₅₀) of KCl cotransport was 60 m in the absence of external K and 3 m at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4 isothiocyanatostilbene-2,2-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4-dinitrostilbene-2,2-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mm. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).This work was supported by grants in aid from the American Heart Association.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

Localization of 5′-ectonucleotidase/phosphatase activity within the olfactory sensilla of the spiny lobster,Panulirus argus

Summary Previous electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has external chemoreceptors, which are selectively stimulated by adenosine 5-monophosphate (AMP) when present in seawater. Subsequent biochemical investigations revealed that AMP can be rapidly dephosphorylated by 5-ectonucleotidase/phosphatase activity associated with the olfactory sensilla (aesthetascs). In this study the deposition of cerium phosphate was used to examine the ultrastructural distribution of 5-ectonucleotidase/phosphatase activity in aesthetascs. Utilizing AMP as substrate, we found dephosphorylating activity to be associated with the outer membranes of both dendrites and auxiliary cells. Moreover, this activity was specifically localized to a narrow band that approximately corresponds to the transitional zone where dendrites develop cilia and branch extensively to form the outer dendritic segments. A similar distribution of the cerium phosphate reaction product was found when -glycerol phosphate was substituted for AMP. The alkaline-phosphatase inhibitor, levamisole, had no apparent effect on the deposition of reaction product when either AMP or -glycerol phosphate was used as substrate. The ectoenzymatic activity in the transitional zone may be of importance in clearing exogenous chemoexcitatory nucleotides from this region.Abbreviations ADP adenosine 5-diphosphate - AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - EM electron microscopy     

Pyridoxal 5′-phosphate levels in brain after treatments which impair cerebral glucose metabolism

Pyridoxal 5-phosphate (PLP) concentrations were measured in brains of rats to determine whether a deficiency of this coenzyme was a common feature in hepatic coma, ethanol intoxication, and in animals treated withl-dopa or with 5-hydroxytryptophan (5-HTP) alone or with inhibitors of MAO or ofl-aromatic amino acid decarboxylase. These treatments have been shown previously to be associated with reduced conversion of glucose to amino acids in brain. Cerebral PLP concentrations were reduced after some of these treatments, notably injection of ethanol, orl-dopa alone or with -phenylisopropylhydrazine, an inhibitor of MAO, or of 5-HTP together withN-[-(chlorophenoxy)ethyl]cyclopropylamine hydrochloride, Lilly 51641, another MAO inhibitor. However, in other circumstances where inhibition of conversion of glucose to amino acids has been shown {treatment with 5-HTP, or with Lilly 51641 or with [N-(d,l-seryl)-N-2,3,4-trihydroxybenzyl]hydrazine, an inhibitor ofl-aromatic amino acid decarboxylase, together withl-dopa or with 5-HTP}, PLP levels in brain were unchanged, or were increased (in hepatectomized rats).     

The synthesis of yellow tetrazolium

Summary The syntheses of 4,4-biphenyl tetrazonium chloride monohydrate, of 1,1-di-(3-nitrophenyl)-3,3-dimethyl-5,5-(4,4-biphenylene)-diformazan, and of yellow tetrazolium [2,2-di-(3-nitrophenyl)-5,5-dimethyl-3,3-(4,4-biphenylene)-ditetrazolium chloride] are described. Solutions of the pure formazan in pyridine absorb visible light maximally at 455 nm; ₄₅₅ is 54,050. Yellow tetrazolium has been specifically designed to meet the requirement of a new technique in quantitative cytochemistry, which involves the consecutive use of two bistetrazolium salts on the same tissue section.I thank Professor L. Young of St. Thomas's Hospital Medical School and Professor C. Long of the Royal College of Surgeons for their interest and encouragement. I am greatly indebted to a large number of friends and colleagues for helpful discussion, and to Dr. A. Deavin and Miss A. Banks for exploratory experiments which involved column chromatography. Microanalyses were carried out by Mr. G. S. Crouch at the London School of Pharmacy.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.