Reactivation by copper of phenolase pre-inactivated by oxalate

The activity of spinach chloroplast phenolase which had been repressed by ammonium oxalate was restored by adding copper. Oxalate appears to bind to the enzyme at a single site, the binding paralleling the inhibition produced at neutral pH. The inhibition of oxalate is due to its binding with copper at the active centre to form an inactive complex, the oxalate moiety of which is releasable when more copper is added. Similar reactivation by copper was obtained with pure mushroom phenolase.     

Association by 2,3-dihydroxybenzaldehyde of monomeric phenolase in spinach chloroplasts

In the presence of 5 mM 2,3-dihydroxybenzaldehyde, the monomeric phenolase (MW 36000) of spinach chloroplasts is completely converted to its dimer within 6 hr without significant change in activity. The aldehyde at concentrations higher than 0.25 mM could bring about this conversion after 18 hr treatment. The association of the two monomers becomes tighter with increasing concentration of the aldehyde. The dimer gave rise to a higher MW protein after freezing briefly. Several mono- and dihydroxybenzaldehydes, 2,3-dihydroxybenzoic acid, and o-vanillin did not produce the dimer.     

Development of an ELISA for estimation of the copper nutritional status of wheat and cotton

Studies were carried out with hydroponically grown wheat and cotton to develop the Cu-requiring protein phenolase as a biomarker of Cu nutrient status. Isozymes of phenolase whose levels were reduced by Cu deficiency were identified by Western blots. A competitive enzyme-linked, immunosorbent assay (ELISA) was developed that could detect as little as 25 ng of phenolase. The ELISA revealed that Cu-sufficient cotton leaves had about 4-fold more phenolase antigen than did Cu-sufficient wheat leaves. In both species, the level of phenolase was reduced by 2- to 5-fold in leaves of Cu-deficient plants. Because the immunoassay for phenolase protein is rapid, inexpensive, and can be carried out with small amounts of leaf material, it has potential as a tool for assessment of the Cu status of crop plants.Abbreviations ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - TBS Trisbuffered saline (20 mM Tris-HCl, pH 9.5, 150 mM NaCl) - TBST Tris-buffered saline containing 0.05% Tween-20     

Phenolase of spinach roots

Phenolase activity is not found in germinating spinach embryos, but it appears in the radicles when the vascular tissues have developed, and then increases progressively. Unlike the two phenolases detected earlier in the chloroplasts, the root enzyme is a single protein with higher MW occurring both in 3000 g precipitate and 28 000 g supernatant fractions. The phenolase in 3000 g fraction is not activated by treatment with detergents and trypsin. The enzyme is contained mainly in xylem parenchymatous cells adjacent to primary vessels. It also occurs to a lesser degree in the dermal parts, including epidermis and cortex. Similar tissue-level distribution patterns of this enzyme are also observed in the roots of other angiosperms, especially in Compositae.     

Inhibition by oxalates of spinach chloroplast phenolase in unfrozen and frozen states

Spinach chloroplast phenolase was inhibited by oxalic acid and its salts. Complete inhibitions were induced instantly in the acidic region (e.g. by 1 and 5 mM oxalate at pH 5 and 5.5, respectively), and in the neutral region pre-incubation of the enzyme with oxalates could also lead to complete loss of activity. The inhibition mode was non-competitive for phenol substrate with K_i of 0.9 mM pH 6.8. Reduction of enzyme activity in a crude extract of chloroplasts induced by freezing at neutral pH was due to the presence of ammonium oxalate. With 0.5 mM oxalate, the inhibition attained 75% under frozen conditions, whilst no inhibition could be detected in the enzyme which had not been frozen. Free oxalic acid and K⁺ and Na⁺ salts also caused freezing inhibition. Glyoxylic and oxamic acids acted as inhibitors with less efficiency. With a pure mushroom tyrosinase (phenolase), essentially the identical results were obtained using the same conditions.     

Effect of sequestering and reducing agents on stabilization of plant dehydrogenase activity in crude extracts

Initial and long-term loss of dehydrogenase activity in crude extracts of herbaceous, and, especially, of woody tissue occur partially because of the inhibitory influence of phenolics. In addition, oxidation of phenols by phenolase results in subsequent enzyme oxidation. Preparation of crude extracts with insoluble PVP, in comparison with anion exchange resins or celluloses, best decreases phenolic concentrations and least decreases dehydrogenase activity in crude extracts. However, removal of phenolics during tissue homogenation does not maximize dehydrogenase activity. Therefore, other methods must be used to stabilize dehydrogenase activity. Sodium ethylenediaminetetracetic acid or sodium azide promoted activity of both purified mushroom and crude plant phenolases. Quinone reduction with diethyldithiocarbamate (DIECA) or mercaptoethanol eliminated apparent phenolase activity, but DIECA inhibited dehydrogenase activity. Elevated concentrations of EtSH diminished initial decay of dehydrogenase activity. Combined use of EtSH and insoluble PVP further stabilized 6-phosphogluconate, glucose 6-phosphate, and malate dehydrogenase, but not glyceraldehyde 3-phosphate dehydrogenase.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Rapid Isolation, Native Enzyme Analysis, Identification of a Serum-Soluble Activity, and Kinetics

The enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.     

Separation of the formation of γ-coniceine and aliphatic amines from got activity in Conium maculatum

L-Alanine:5-keto-octanal aminotransferase has been separated from GOT and GPT activities by DEAE-cellulose chromatography. Two transaminase peaks A and B were observed for γ-coniceine formation and assay showed that these peaks also contained the amino acid:aldehyde aminotransferase (AAT) responsible for aliphatic amine biosynthesis. Kinetic studies showed that γ-coniceine formation with transaminase A had K_{m for L-alanine and 5-keto-octanal of 27 mM and 1.6 mM respectively. The Vmaxwas 1.3 nkats/mg protein and the activation energy was 3.0 kJ/mol. For transaminase B the Kinm} for L-alanine and 5-keto-octanal are 55 mM and 0.14 mM respectively with a V_maxof 3.3 nkats/mg protein and an activation energy of 0.33 kJ/mol. Transaminase A and B had the same MW and have distinguishable pH_max. γ-Coniceine formation by transaminase A was inhibited uncompetitively by glyoxalate and competitively by pyruvate, whereas with transaminase B uncompetitive inhibition was also observed with glyoxalate; no inhibition was observed with pyruvate which at low concentration (0.25 M) showed slight stimulation of activity.     

Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activator activity.

A functionally active human plasmin light (B) chain derivative, stabilized by the streptomyces plasmin inhibitor leupeptin, was isolated from a partially reduced and alkylated enzyme preparation by an affinity chromatography method with a L-lysine-substituted Sepharose column. This light (B) chain derivative was found to be relatively homogeneous by electrophoretic analysis in both an acrylamide gel/dodecyl sulfate system and on cellulose acetate. It possessed approximately 3% of the proteolytic activity (casein substrate) of the original enzyme, and it incorporated 0.09 mol of [3H]diisopropyl phosphorofluoridate per mol of protein. It contained 3.1 +/- 0.3 carboxymethylated cysteines per mol of protein and can be designated as a CmCys5-light (B) chain (CmCys)3. When this isolated light (B) chain derivative was mixed in equal molar amounts with streptokinase, the mixture developed both human and bovine plasminogen activator activities; the bovine activator activity was approximately 66% of the bovine activator activity of the equimolar human plasmin-streptokinase complex. Although this complex now incorporated 0.50 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was the same as the proteolytic activity of the isolated light (B) chain derivative. It was shown by electrophoretic analysis in both an acrylamide gel/epsilon-aminocaproic acid system and on cellulose acetate that the light (B) chain derivative and streptokinase forms an equimolar light (B) chain-streptokinase complex, indicating that the binding site for streptokinase is located on the light (B) chain of the enzyme. A functionally active equimolar light (B) chain-streptokinase complex was also isolated from a partially reduced and alkylated equimolar human plasmin-streptokinase complex by the affinity chromatography method. The plasminogen activator activities (human and bovine) of this light (B) chain-streptokinase complex were similar to those of the plasmin-streptokinase complex from which it was derived. Although this complex incorporated 0.70 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was only 14% of proteolytic activity of the plasmin-streptokinase complex.     

Arginine Decarboxylase of oat seedlings

Arginine decarboxylase activity in the shoots of seedlings was high in oats, intermediate in barley and low in rice, maize, wheat and rye. After partial purification, the arginine decarboxylase from the shoots of potassium deficient oat seedlings was separated into two fractions, A (MW 195 000) and B (MW 118 000), by gel chromatography. On gel electrophoresis, the mobilities of these fractions were respectively 0.12 and 0.55 relative to bromophenol blue at pH 9.5. Fraction A was twice as active as fraction B in extracts of seedlings grown with both normal and potassium deficient nutrition, despite the greater activity ( × 5) of the potassium deficient plants. The properties of the two fractions were similar with respect to pH optimum (7–7.5), K_m (3 × 10 ^?5M) and the effect of inhibitors. Fraction A was purified to apparent homogeneity by DEAE-cellulose chromatography. The enzyme was specific for l-arginine and it was strongly inhibited by NSD 1055, d-arginine and canavanine. Mercaptoethanol and dithiothreitol stimulated the enzyme by ca 50% and p-chloromercuribenzoate was an inhibitor. Pyridoxal phosphate stimulated activity by ca 30% and EDTA stimulated activity by 30%. Ca²⁺ and Mg²⁺ inhibited the enzyme by 50% at ca 20 mM. Putrescine and the polyamines showed only moderate inhibition at 10 mM, but agmatine reduced activity to 30% at this concentration.     

Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.

Modulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80,000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulator-deficient myosin light-chain kinase (MW 80,000), modulator protein (MW 16,500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10 mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin--tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28 mM and 0.024 mM, respectively. Vm/e was 5.7 s-1.     

淡色库蚊中抗性相关羧酸酯酶的纯化及其生化性质

在库蚊Culex pipiens品系中,非专一性酯酶活性的升高是对有机磷杀虫剂产生抗性的重要机理之一。应用SDS/PAGE,比较淡色库蚊Culex pipiens pallens抗敌百虫品系(RD)、敏感型品系(S)和抗苄呋菊酯品系(PY)中可溶性总蛋白质带型,显示RD中含有一条特异蛋白带,其它两个品系中未检出。在RD成虫匀浆液总蛋白中含量高达2.1%。分子量测定为66 kD。应用柱层析法分离得到了较纯的纯品。以α-NA为底物测得K_m=64.1 mmol/L,V_max=249.8 mmol/(L·mg·min)。与羧酸酯酶相比较：其K_m值小于已报道的抗性品系及非抗性品系A-酯酶和B-酯酶。V_max值比已报道抗性品系A-酯酶低,比B-酯酶高。较高浓度的敌百虫并不能抑制其酶活,属于A-酯酶。在昆虫体内可能主要通过结合隔离作用(sequestration)提高昆虫对有机磷的耐受性,对有机磷杀虫剂水解作用的可能性也不能排除。     

Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit.

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.     

Activation of latent phenolase during spinach leaf senescence

The observed increase of phenolase activity and of its rate of activation during spinach leaf senescence is due to reduced binding of latent phenolase to the thylakoid membranes and not to de novo synthesis. The same amount of phenolase which is active in isolated thylakoid membranes from senescent leaves can be found in the membranes of non-senescent leaves after activation of latent enzyme. Tracer experiments give evidence that one multiple form which is responsible for the bulk activity in senescent leaves, is synthesized before, but not after the onset of senescence, indicating that pre-existing latent phenolase is converted to easily activating forms.     

o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro

A human follicular fluid (HFF) fraction prepared by Sephadex G-75 column chromatography has been previously shown by this laboratory to initiate the human sperm acrosome reaction (AR) in vitro. In the present report, the apparent molecular weight (MW) of this AR activity determined by a longer G-75 column than was used in the previous work was 50,000 ± 5,106. The G-75 Sephadex void volume fractions of some but not all HFF samples were also found to contain some AR-initiating activity. The occasional void volume activity was less potent than that of the 50,000 MW fraction and was not studied further. Further characterization of the 50,000 MW fraction was carried out. A time-course study demonstrated that maximum AR were obtained within 5 min following the addition of the 50,000 MW fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining revealed that the 50,000 MW fraction was still a relatively crude preparation. Treatment of the 50,000 MW fraction with chloroform:methanol did not extract the AR-initiating activity into the lipid phase. The AR-initiating activity of the untreated 50,000 MW fraction was precipitated when it was boiled, but the activity was partially resistant to boiling after overnight incubation. Treatment of the 50,000 MW fraction with pronase E or with several glycosaminoglycan hydrolases did not destroy the activity. Pronase treatment resulted in a higher amount of boiling-resistant AR-initiating activity. The AR-initiating activity of the untreated 50,000 MW fraction was partially dialyzable, but the activity of an undialyzed fraction did not pass through an ultrafiltration membrane with a 10,000 MW cut-off. However, treatment of the 50,000 MW fraction with protease, peptide:N-glycosidase F, and to a lesser extent chondroitinase ABC yielded an active lower MW activity which could pass through such an ultrafiltration membrane. The lower MW activity released by peptide:N-glycosidase F eluted in the included volume (5,000–1,000) of a Sephadex G-25 column. Neutral hexose but not protein or peptide was detected in the G-25 peak of AR-initiating activity. These results suggest that the AR-initiating activity present in the 50,000 MW fraction of HFF: (1) is present either as two different AR factors (a high-MW factor and a low-MW, noncovalently bound factor) or as a single factor responsible for both the nondialyzable and dialyzable AR-initiating activities (the latter being enzymatically released from the former), and (2) may be at least partially associated with N-linked oligosaccharides of a glycoprotein or proteoglycan.     

Construction of the bifunctional enzyme cellulase-β-glucosidase from the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

An artificial bifunctional enzyme, cellulase-β-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and β-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or β-glucosidase. The fusion enzyme was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70°C over 30 min, and optimal β-glucosidase activity at pH 7.0 and 80°C over 30 min.     

Oxidative enzymes in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The activities of phenolase, peroxidase, cytochrome oxidase, catalase and superoxide dismutase, as well as the levels of lipid peroxides, were measured in plerocercoids of S. solidus taken from the body cavity of the fish (unactivated) and in plerocercoids which had been cultured in vitro, either under air, or under 95% N₂, 5% CO₂. When cultured anaerobically, the activities of phenolase, peroxidase and cytochrome oxidase all increased dramatically. Aerobically, only phenolase activity increased. Lipid peroxide levels and superoxide dismutase activity was similar at all stages and catalase could not be detected. It is suggested that the increased activity of oxidative enzymes in anaerobically cultured worms is an attempt to compensate for the reduced environmental pO₂.     

Design of human granzyme B variants resistant to serpin B9

Human granzyme B (hGB) is a serine protease involved in immune‐mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine‐scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild‐type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild‐type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Purification of human interleukin 1

Interleukin I (IL-1) is a lymphocyte stimulant released by human monocytes cultured for 18–24 hours in tissue culture medium containing 5% serum and the non-specific immunostimulant lipopolysaccharide (LPS). Human IL-1 is found in the conditioned medium in a low molecular weight (～ 13,000) and a high molecular weight (～ 85,000) form. The high MW activity may result from the formation of a complex between IL-1 and serum constituents. During the course of purification, the low MW IL-1 activity is often recovered in a high MW form. Hollow fiber diafiltration and membrane ultrafiltration has been found to rapidly separate low MW IL-1 from all measurable protein with a yield of 4% of the original activity. The IL-1 which converts to the high MW form during the purification is recoverable, 21% of the original activity, but contains small amounts of serum proteins. Isoelectric focusing (IEF) of the low MW IL-1 resulted in a very highly purified sample which was analyzed by polyacrylamide gel electrophoresis (PAGE). Utilizing a new staining procedure which detects less than 1 ng of protein per band, the IEF-purified IL-1 revealed trace quantities ( < 1 ng) of a slowly migrating protein similar to immunoglobulin and no other bands. There were no bands which corresponded with the known electrophoretic mobility of IL-1. Since the samples applied to the gel contained significant biological activity, this result implies that human IL-1 is biologically active in picogram quantities.