Eimeria tenella: specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol.

t-Butylaminoethanol is an anticoccidial compound that is related structurally to the metabolically active substances, dimethylaminoethanol, and choline. Toxic effects of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylamino-ethanol): metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:10 over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:Dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1:7 with a concentration of 0.015% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor.     

Characterization of a lactate dehydrogenase inhibitor protein from rabbit skeletal muscle mitochondrial fraction

A lactate dehydrogenase inhibitor protein is isolated from rabbit skeletal muscle crude mitochondrial fraction. The molecular weight of the inhibitor is approximately 20,000 as determined by size exclusion HPLC. The inhibitor isoelectricpH is 5.3 as determined by agarose or polyacrylamide gel isoelectric focusing. The amino acid composition of the inhibitor is given. The presence of the inhibitor gives an acidic characteristic to the alkaline M₄ lactate dehydrogenase isozyme and the lactate dehydrogenase-inhibitor complex is more stable than the enzyme alone.     

A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein

A thermostable NAD(P)⁺ glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.     

Hemin control of globin synthesis: an assay for the inhibitor formed in the absence of hemin and some characteristics of its formation

In the absence of hemin, an inhibitor of globin chain initiation is formed in the ribosome-free supernatant fraction of rabbit reticulocytes. The inhibitor may be assayed in a hemoglobin-synthesizing system containing the complete lysate and hemin. The potency of a unit of inhibitor is not altered over a broad hemin concentration range. A lag phase in inhibitor formation of two and four hours occurs if the supernatant fraction is incubated at 25 °C and 20 °C respectively. Although hemin almost completely suppresses formation of inhibitor if present from the beginning of an incubation, it only partially suppresses formation if added at the end of the lag phase. This is interpreted as indicating the presence of an intermediate in the conversion of a pro-inhibitor to inhibitor. Hemin blocks the formation of the intermediate but not its conversion to inhibitor. The formation of inhibitor continues for eight to twelve hours at 34 °C but stops in one hour at 50 °C, suggesting the possible participation of a heat labile factor. This is supported by the observation that a mixture of two supernatant fractions, one blocked in inhibitor formation with hemin and the other by heating at 50 °C, can form additional inhibitor at 37 °C.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

The control of aldolase in Lemna minor L. in relation to nitrogen deficiency

The loss of activity of aldolase which occurs when Lemna is deprived of nitrogen is shown to be due to the accumulation of a specific inhibitor of aldolase. The inhibitor has been purified 600-fold and has the properties of a low molecular weight protein. The inhibitor is not a proteolytic enzyme and the kinetics of the interaction between aldolase and the inhibitor are reported. The possible physiolgocal significance of the inhibition of aldolase is briefly discussed.     

Purification of a high-affinity discoidin I-binding proteoglycan from axenic Dictyostelium discoideum growth medium

The axenic Dictyostelium discoideum growth medium HL-5, prepared using Difco proteose peptone No. 2, contains an extremely potent inhibitor of the binding of ¹²⁵I-labeled discoidin I to glutaraldehyde-fixed, cohesive D. discoideum cells. Axenic strain A3 D. discoideum cells bind or internalize the inhibitor during growth in HL-5 medium and subsequently shed or excrete it while differentiating in suspension. The inhibitor has been purified from Difco proteose peptone No. 2 by sequential gel filtration on Sepharose 4B and affinity adsorption using discoidin I-Sepharose. The inhibitor is heterogeneous in molecular weight (4 · 10⁵?2 · 10⁶), but is relatively homogeneous in density on CsCl density gradients. The size and activity of the inhibitor are resistant to periodate, reduction and maleylation, proteases, nucleases and heating in the absence or presence of sodium dodecyl sulfate. Mild alkali causes a partial reduction in activity and converts the higher molecular weight fraction of the inhibitor to a lower molecular weight. The purified inhibitor contains neutral hexose, hexosamine and amino acid in an approximate molar ratio of 4 : 3 : 2. These and other properties suggest that the inhibitor is an unusual proteoglycan. Certain well-characterized glycosaminoglycans are relatively potent inhibitors of discoidin I binding. The proteoglycan reported here is the most potent discoidin I-binding inhibitor ever identified.     

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F → E → C → A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

A Protease Inhibitor from Penicillium cyclopium

A protease inhibitor produced by Penicillium cyclopium on solid cultures of wheat bran was purified by means of column chromatography on Duolite A-2 and DEAE-cellulose, acetone precipitation and lyophilization. The purified inhibitor obtained as a white, floccose and hygroscopic substance was monodisperse by ultracentrifugal analysis. It was found to be an acidic macro-molecule of a molecular weight of about 5000. The chemical analyses rejected the possibility of the presence of amino acids, peptides, sugars, amino sugars, or uronic acids in the inhibitor molecule.

Properties of a protease inhibitor from Penicillium cyclopium were studied. The pH range of the inhibitor action is restricted to acid pH, optimally at pH 3. Increasing temperature accelerates its action upon enzyme. The inhibitor causes enzyme inactivation in proportion to its concentration. It is fairly stable in an acid solution but unstable in an alkaline solution. It undergoes destruction by heat, hydrogen peroxide and ascorbic acid. The inhibitor reversibly combines with Al³⁺, Fe³⁺, Ag⁺ and Cu²⁺ to produce a precipitate. Salts interfer with the inhibitor activity. Generally, acid proteases from various penicillia are susceptible to the inhibitor while those from other genera are resistant.     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

Isolation and properties of an inhibitor of phospholipase A from Bothrops neuwiedii venom

An inhibitor of phoapholipase A has been isolated from Bothrops neuwiedii venom after gel filtrations through Sephadex G-50 (pH 4.5), Sephadex G-25 (pH 7.6), Sephadex G-15 (pH 4.0), and chromatography on SE-Sephadex C-25 (pH 4.2–4.5). When subjected to paper electrophoresis, the inhibitor migrates as a simple compound with isoelectric point near pH 6.8. Aminoacid composition, sensitivity toward proteases, and the absorption spectrum fit in well with a polypeptide structure lacking tyrosine and tryptophan. In the absence of EDTA, an inactive, anionic derivative appears in inhibitor preparations; the reaction can be reversed by 2-mercaptoethanol. Direct interaction of enzyme and inhibitor is proved by the inhibition of enzyme activity and the chromatography of enzyme-inhibitor mixtures. Titration of inhibitor with venom phospholipases A (isoenzymes P-1 and P-2) yields sigmoid-shaped concentration-inhibition curves, with P-1 far more sensitive than P-2. The enzyme-inhibitor interaction depends on pH since it is tight at pH 4.5 but does not occur at pH 7.5. Presence of thiol groups in inhibitor is consistent with (a) characteristic spectral changes after reaction of inhibitor with PMB ⁴ and NEM; (b) the inhibitor inhibition by PMB, NEM, iodoacetate, and Hg²⁺, and (c) the reversal of PMB inhibition with reduced glutathione. Since phospholipase A is insensitive towards Hg²⁺, addition of Hg²⁺ to enzyme-inhibitor mixtures (or crude venom samples) causes an apparent enzyme activation (deinhibition). When substrate (egg-yolk lipoprotein) is added to enzyme-inhibitor mixtures, the reaction kinetics show an initial “lag-period” which is proportional to the inhibitor concentration. The “lag-period” does not occur in the absence of inhibitor or in the presence of Hg²⁺, that inactivates the inhibitor.     

Control of growth by auxin and its specific neutral inhibitor

Evidence for the existence of a neutral inhibitor specific for auxin has been in the literature for 45 years. The inhibitor is demonstrable by its effect in causing positive curvature of theAvena coleoptile. The growth of the mesocotyl of oat and corn seedlings in darkness and its inhibition by light are determined by the neutral inhibitor as is the phototropic response of the sunflower stem. Production of the inhibitor is promoted by red and fluorescent light. Irradiance at 730 nm promotes auxin production while irradiance at 660 nm promotes production of the inhibitor. The positive curvature induced by 2,3,5-triiodobenzoic acid can be used to quantify the neutral inhibitor. Since the benzoic acid is more effective than iodoacetate in reacting with the sulfhydryl of cysteine, a sulfhydryl group is indicated to bo one reaction site for auxin and to be the basis of polar transport.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency     

Metalloenzyme inhibitor from kidney beans: partial purification and characterization

Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn²⁺. One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn²⁺. The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺. The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn²⁺ from the enzymes.     

Detection, Purification and Identification of An Endogenous Inhibitor of l-Dopa Decarboxylase Activity from Human Placenta

An endogenous inhibitor of l-Dopa decarboxylase activity was identified and purified from human placenta. The endogenous inhibitor of l-Dopa decarboxylase (Ddc) was localized in the membrane fraction of placental tissue. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K did not affect membrane-associated Ddc inhibitory activity, suggesting that a population of the inhibitor is embedded within membranes. Purification was achieved by extraction from a nondenaturing polyacrylamide gel. The purification scheme resulted in the isolation of a single 35 kDa band, bearing l-Dopa decarboxylase inhibitory activity. The purified inhibitor was identified as Annexin V. The elucidation of the biological importance of the presence of an l-Dopa decarboxylase activity inhibitor in normal human tissues could provide us with new information leading to the better understanding of the biological pathways that Ddc is involved in.