Proteinaceous inhibitor versus fructose as modulators of Pteris deflexa invertase activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer Mr 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had beta-fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with Km of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

Simultaneous interaction of enzymes with two modifiers: Reappraisal of kinetic models and new paradigms

Enzyme activity can be modulated by the concurrent action of two modifiers, either activators or inhibitors. The kinetic mechanisms for the interaction of the individual modifiers with the target enzyme can change considerably when two modifiers bind simultaneously. We illustrate a general equation for this kind of interactions, which can unambiguously describe the behavior of activators and inhibitors acting by any combination of classical kinetic mechanisms. The flexibility of this model is exemplified by combinations of activators and/or inhibitors, which can be competitive, uncompetitive or mixed-type, bind the target enzyme in either compulsory or random order, and are able to drive or not enzyme activity to zero at saturation. The model shows that the effects of zero-interaction and synergy between simultaneously acting enzyme modifiers are common events. Yet, in disagreement with previous theories, this model shows that antagonism between enzyme modifiers is a rare effect, which can be predicted only under very particular circumstances.     

Effects of Oxodiperoxovanadate (V) Complexes on the Activity of Green Crab (Scylla serrata) Alkaline Phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in seawater on the activity of the enzyme will result in the loss of the biological function of the enzyme, which will affect the exuviating crab shell and threaten the survival of the animal. In the present paper, the effects of four oxodiperoxovanadate (V) complexes on the activity of green crab alkaline phosphatase have been studied. The results show that these vanadate derivatives can lead to reversible inactivation. The equilibrium constants for binding of inhibitors with the enzyme and/or the enzyme–substrate complexes have been determined. The results show that sodium (2,2'-bipyridine)oxodiperoxovanadate, pV(bipy), and potassium oxodiperoxo-(1,10-phenanthroline)vanadate, pV(phen), are competitive inhibitors, while potassium picolinato-oxodiperoxo-vanadate, pV(pic), and oxalato-oxodiperoxovanadate, pV(ox), are mixed-type inhibitors. These results suggest that pV(bipy) is a considerably more potent competitive inhibitor than pV(phen) and that the competitive inhibition effect of pV(pic) is stronger than that of pV(ox), but the non-competitive inhibition effect of pV(ox) is stronger than that of pV(pic).     

Lysis of tumor cells by antibody and complement. VI. Enhanced killing of enzyme-pretreated tumor cells.

The ascites form of a chemically induced guinea pig hepatoma, line-10, was resistant to killing in vitro by xenogeneic antibody and guinea pig complement. Pretreatment of line-10 cells with certain proteolytic enzymes rendered tham susceptible to the killing action of antibody and guinea pig complement. The effects of enzyme pretreatment were dependent on enzyme concentration, temperature, and could be blocked by addition of competitive or non-competitive inhibitors. The effect of the enzyme treatment could reversed by incubating the treated cells at 37 degrees C (but not at 0 degrees C), in the absence of the enzyme. Effective enzymes included ficin, bromelain, pronase, elastase, papain, trypsin, collagenase, lipases type I and type VI, and the neuraminidase preparation isolated from Clostridium perfringens. The activity of the lipase preparations and the neuraminidase preparation isolated from Clostridium perfringens appeared to be caused by proteolytic enzyme contamination. Enzyme preparations that proved ineffecitve in rendering the line-10 cells sensitive to killing by antibody and guinea pig complement included DNase, RNase, beta-glucuronidase type 6A or type B10, hyaluronidase type V or type VI, and pectinesterase.     

Purification, characterization and cellular localization of 5''-nucleotidase from Torpedo electric organ.

5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.     

Modulation of latent protein phosphatase activity from vascular smooth muscle by histone-H1 and polylysine

An apparently latent phosphatase which migrated as a protein of Mr 130,000 during sucrose density centrifugation, and a spontaneously active phosphatase (Mr 68,000) were isolated from bovine aortic smooth muscle. Basal phosphorylase phosphatase activity of the latent preparations was stimulated 12 fold by low concentrations of lysine-rich histone-H1 (30 micrograms/ml) and 6 fold by polylysine (Mr 17,000; 12 micrograms/ml), whereas the spontaneously active enzyme was only slightly affected. The enzymatic activity of the spontaneously active preparation was completely destroyed by beta-mercaptoethanol. In contrast, the apparently latent enzyme was converted to a more active form of lower molecular weight (Mr 86,000) following treatment with beta-mercaptoethanol and this form of the enzyme was still stimulateable by histone-H1. These findings show that the aortic spontaneous and apparently latent phosphatase actives are ascribable to separate enzymes and they suggest that the activity of latent phosphatase in living cells may be modulated by cationic proteins such as histones or similar effector molecules.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.     

Novel properties of human monocyte plasminogen activator

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.     

Inhibition of glutamine synthetase activity by biologically active derivatives of glutamic acid

The inhibition of activity of glutamine synthetase from Chlorella and porcine brain by 4-hydroxy-D-4-fluoro-D,L- and 4-amino-D,L-glutamic acids diastereoisomers was studied. Each compound was shown to exert the same inhibiting effect on glutamine synthetase from both sources. In case of threo-4-hydroxy-D-glutamic acid the inhibition of the Chlorella enzyme was of a competitive and of a completely mixed type. The enzyme inhibition by 4-fluoro-D, L-glutamic acids seemed to be of a completely non-competitive type. The Ki values for all inhibition reactions were determined. A comparison of biochemical parameters and biological activity revealed that the most effective inhibitors of the enzyme exert a most potent antitumour and antiviral action.     

Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma.

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.     

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.     

NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP⁺) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M_r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.     

Proteinaceous Inhibitor Versus Fructose as Modulators of Pteris deflexa Invertase Activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer M r 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had β -fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with K m of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

Ubiquitin-based anticancer therapy: Carpet bombing with proteasome inhibitors vs surgical strikes with E1, E2, E3, or DUB inhibitors

The proteasome inhibitor bortezomib remains the only ubiquitin pathway effector to become a drug (VELCADE?) and has become a successful treatment for hematological malignancies. While producing a global cellular effect, proteasome inhibitors have not triggered the catastrophe articulated initially in terms such as "buildup of cellular garbage". Proteasome inhibitors, in fact, do have a therapeutic window, although in the case of the prototype bortezomib it is small owing to peripheral neuropathy, myelosuppression and, as recently reported, cardiotoxicity [1]. Currently, several second-generation molecules are undergoing clinical evaluation to increase this window. An alternative strategy is to target ubiquitin pathway enzymes acting at non-proteasomal sites-E1, E2, and E3, associated with ubiquitin conjugation, and deubiquitylating enzymes ("DUBs")-that act locally on selected targets rather than on the whole cell. Inhibitors (or activators, in some cases) of these enzymes should be developable as selective antitumor agents with toxicity profiles superior to that of bortezomib. Various therapeutic hypotheses follow from known cellular mechanisms of these target enzymes; most hypotheses relate to cancer, reminiscent of the FDA-approved protein kinase inhibitors now marketed. Since ubiquitin tagging controls the cellular content, activity, or compartmentation of proteins associated with disease, inhibitors or activators of ubiquitin conjugation or deconjugation are predicted to have an impact on disease. For practical and empirical reasons, inhibitors of ubiquitin pathway enzymes have been the favored therapeutic avenue. In approximately the time that has elapsed since the approval of bortezomib in 2003, there has been some progress in developing potential anticancer drugs that target various ubiquitin pathway enzymes. An E1 inhibitor and inhibitors of E3 are now in clinical trial, with some objective responses reported. Appropriate assays and/or rational design may uncover improved inhibitors of these enzymes, as well as E2 and DUBs, for further development. Presently, it should become clear whether one or both of the two general strategies for ubiquitin-based drug discovery will lead to truly superior new medicines for cancer and other diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Environmental and chemical dissociation of antibody-dependent phagocytosis from lysis mediated by macrophages: Stimulation of lysis by sulfhydryl-blocking and esterase-inhibiting agents and depression by trypan blue and trypsin

Peritoneal exudate cells and RAW264 macrophage tumor culture line were tested for antibody-dependent effector activity to sheep red blood cells (RBC). Assay in tissue culture dishes showed mostly lysis of targets while tubes promoted phagocytosis. The kinetics suggested that these were independent processes. At concentrations inhibiting ingestion, sulfhydryl-blocking agents iodoacetate, N-ethylmaleimide, and p-chloromercuribenzoate, and esterase inhibitors tosyl-lysine chloromethyl ketone, and diisopropyl fluorophosphate stimulated lysis of RBC. Pretreatment of effector cells but not targets also augmented the lytic reaction. Three other macrophage cell lines with very weak killing activity were stimulated by iodoacetate, but two lymphoid cell lines showed no cytotoxicity with or without the drug. In contrast to these enzyme inhibitors, trypan blue blocked peritoneal exudate and cell line lysis with no effect on phagocytosis. Trypsin pretreatment also inhibited RAW264 but not peritoneal cell cytotoxicity. There was little effect of these agents on macrophage Fc receptors as measured by EA rosettes, or on cell viability or production of lysozyme and β-glucuronidase. We conclude that the two macrophage effector mechanisms are independent, can be inversely modulated by environmental conditions (assay vessel), and are regulated by enzymes or proteins specific for each process.     

Inhibition of microbial cholesterol oxidases by dimethylmorpholines

Cholesterol oxidase is a potentially important enzyme in steroid transformations, catalysing the conversion of 3-hydroxy-5-ene steroids to 3-keto-4-ene derivatives via a 3-keto-5-ene intermediate. Morpholine derivatives, especially fenpropimorph and tridemorph, were found to block selectively the isomerisation activity of cholesterol oxidases isolated from Nocardia erythropolis, Streptomyces sp., Pseudomonas testosteroni and Schizophyllum commune. These enzymes differ strongly in physical characteristics and catalytic behaviour. The effectiveness of the inhibitors varied with the cholesterol oxidase tested. Fenpropimorph was most effective with each of the 4 enzymes, 50 mg/l inhibiting about 50% of the enzyme activity. Inhibition was instantaneous and followed a reversible competitive mechanism in Streptomyces sp. and a reversible non-competitive mechanism in Nocardia erythropolis and Schizophyllum commune. An irreversible type of inhibition was observed for P. testosteroni cholesterol oxidase.     

The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.