Immunoadsorbent isolation of trypsin inhibitors from corn and teosinte seeds

An immunoadsorbent was constructed by covalently coupling 100 mg of affinity-purified antibodies against corn trypsin inhibitor to cyanogen bromide-activated agarose. With the immunoadsorbent, we could purify the trypsin inhibitor in aqueous extracts of corn (Zea mays) seeds in a single chromatographic step. The immunoadsorbent was found to have the capacity to bind about 6 mg of corn inhibitor. Recovery of exclusively virgin corn inhibitor required the addition of protease inhibitors to the corn extract. The virgin inhibitor isolated by use of the immunoadsorbent was found to be indistinguishable from the single-chain inhibitor recovered previously from chromatography of corn extracts on trypsin-agarose (Swartz, M.J., Mitchell, H.L., Cox, D.J. and Reeck, G.R. (1977) J. Biol. Chem. 252, 8105–8107). The immunoadsorbent was also used to isolate trypsin inhibitor seeds of teosinte (Zea mexicana), which is believed to be the progenitor of corn.     

Proteinase inhibitors from lonchocarpus capassa (appleleaf) seed

Four proteinase inhibitors (DE-1 to DE-4) were purified from L. capassa seed by chromatographic procedures involving Sephadex G-50 and DEAE-cellulose. They comprise each 80 amino acids (MW ca 10 000) including fourteen half-cystine residues. The partial amino acid sequence of inhibitor DE-4 was determined; 60 of the 80 residues have been sequenced. The MW, cystine content and partial sequence of DE-4 resemble those of the Bowman-Birk-type proteinase inhibitors. The properties of inhibitors DE-1 and DE-4 are very similar. Each contains a potent inhibitor for porcine trypsin but they inhibit bovine α-chymotrypsin only weakly.     

The primary structure of the human pancreatic secretory trypsin inhibitor: Amino acid sequence of the reduced S-aminoethylated protein

The amino acid sequence of human pancreatic secretory trypsin inhibitor [Pubols, M. H., Bartelt, D. C., and Greene, L. J. (1974) J. Biol. Chem., 249, 2235–2242] was determined by a combination of selective trypsin and chymotrypsin hydrolysis reactions on the S-2-aminoethylcysteinyl inhibitor and conventional methods (subtractive Edman degradation and exopeptidase hydrolysis) for sequence determination of small peptides. The peptides were ordered on the basis of the identification of the amino- and carboxyterminal residues of the products at each stage of the degradation procedure. The sequence determination was carried out on a mixture of chromatographic forms present in both tissue and pancreatic juice which are identical in amino acid composition, aminoterminal residues, molecular weight, and specific activity, but differ only in asparagine content and susceptibility to enzymatic hydrolysis. The amino acid sequence of the human inhibitor corresponding to chromatographic form A₃ has been shown to be NH₂

.     

Nature of the Conversion of Ficus carica Variety Kadota Ficin Component D to Component C. Some Physicochemical Properties of Components C and D

Component C can be formed from component D under the experimental conditions used during purification of Ficus carica variety Kadota latex. By use of the inhibitor, sodium p-chloromercuribenzoate, the 2 components have been purified to chromatographic homogeneity. The 2 components have identical molecular weights and amino acid composition. The only difference found between the 2 components is the presence of 3 to 6 more amide groups in component D than in component C. There also appears to be a conformational difference between the 2 since component C is not as acidic, with respect to component D, as would be expected from the comparative amide contents. Conformational differences between the 2 are also indicated by the chromatographic behavior of the 2 in the presence and absence of sodium-p-chloromercuribenzoate.     

A rapid method for the purification of β-lactamase from Bacillus cereus by affinity chromatography

A procedure for the purification of β-lactamase from Bacillus cereus in a single chromatographic step is described. The enzyme is isolated from the crude culture supernatant by affinity chromatography. An inhibitor, methicillin, was immobilised by covalent attachment to the insoluble column gel, Sepharose. The enzyme was adsorbed to the column ligand from the crude supernatant and was subsequently released by increasing the ionic strength of the eluting buffer. In this way the enzyme was selectively isolated from other proteins in the crude supernatant. About 98% of the original β-lactamase activity was recovered in the purified enzyme fraction.     

Characterization and Role of an Endogenous Inhibitor in the Induction of Cold Hardiness in Acer negundo

An inhibitor extracted from short day treated Acer negundo leaves was compared to abscisic acid in 4 different solvent systems. The chromatographic properties of abscisic acid and the inhibitor were in very close agreement. Treatment of Acer negundo plants under non-hardening preconditions (long days) with either the inhibitor or abscisic acid increased hardiness after a hardening period of 3 weeks at 40°. A gibberellin-inhibitor relationship was further studied by making comparison of extracts of plants subjected to either 4 weeks of long days, long days + 5° nights, or short days. These tests indicated that gibberellin-like activity was greatest when the treatment included long days. Abscisic acid-like levels were highest when the treatments consisted of short days or long days + 5° nights. Since the latter groups are the most capable of developing hardiness, the hardening process appears to be more closely related to a build-up of abscisic acid levels than a reduction of gibberellin levels.     

Identification of protein binding partners of ALK-5 kinase inhibitors

We have investigated the binding characteristics of a potent member of the bis-ortho-substituted five-membered nitrogen heterocycle class of ALK-5 kinase inhibitors with lysates of cultured HEK-293 cells to identify protein binding partners of potential biological significance. An affinity chromatographic resin containing an immobilized ALK-5 kinase inhibitor, 2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, was used to capture specific proteins from the cell lysate. The soluble inhibitor was then used to specifically elute the proteins which selectively bound to the pharmacophore ligand structure. Application of 2-D SDS–PAGE analysis with positive and negative controls demonstrated the inhibitor bound several different proteins via selective molecular recognition processes. The structural features of the specifically eluted proteins were identified by peptide mass fingerprinting (PMF) methods and included proteins with structural, metabolic and chaperone functions. Furthermore, these PMF results identified the therapeutic target in various cancer treatment studies, HSP-70, as a potential high-affinity binding partner. These observations warrant examination of bis-ortho-substituted five-membered nitrogen heterocycles as dual ALK-5/HSP-70 inhibitors for anti-cancer drug development.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300–1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35 000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60°C) and resistant to inactivation by trypsin (2 h, 37°C, 10 μg/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.     

Action of 5-thio-D-glucose and its 1-phosphate with hexokinase and phosphoglucomutase.

Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a K_m of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K_i of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K_m of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K_i of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions.     

In silico modeling and docking studies of Soap Nut Trypsin Inhibitor

Soap nut trypsin inhibitor (SNTI), an inhibitory protein was isolated and purified from the seeds of Sapindus trifoliatus by ammonium sulphate precipitation followed by ion-exchange and gel filtration chromatographic techniques and was found to be homogenous by PAGE. Using advanced proteomic techniques like MALDI-TOF the inhibitor was sequenced and analyzed using MASCOT software. A refined 3D model of the structure was predicted by in silico technique like homology modeling. The docked interactions between the predicted structure of SNTI and bovine trypsin were studied using ClusPro 2.0. Further docking results indicate that residues within the receptor binding domain include N145, R241, P242, L243, R244, R249, E266 and R275 respectively which play a key role in protein–protein interaction between SNTI and 3MFJ (bovine trypsin). SNTI was also known to exhibit potent anti-fungal activity against dandruff causing fungi. This study provides an insight into the structure of SNTI and also gives an idea about potential sites responsible for inhibitory action that could further be substantiated by experimental investigations.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD⁺-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.     

Synthesis and Release of Cyclic Adenosine 3':5'-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [³²P]cAMP was purified from cultures supplied [³²P]phosphate. The compound was identified as [³²P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a ³²P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a ³²P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [³H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.     

Methane, Carbon Dioxide, and Hydrogen Sulfide Production from the Terminal Methiol Group of Methionine by Anaerobic Lake Sediments

A significant portion of the sulfide in lake sediments may be derived from sulfur-containing amino acids. Methionine degradation in Lake Mendota (Wisconsin) sediments was studied with gas chromatographic and radiotracer techniques. Temperature optimum and inhibitor studies showed that this process was biological. Methane thiol and dimethyl sulfide were produced in sediments when 1-μmol/ml unlabeled methionine was added. When chloroform (an inhibitor of one-carbon metabolism) was added to the sediments, methane thiol, carbon disulfide, and n-propane thiol were produced, even when no methionine was added. When ³⁵S-labeled methionine was added to the sediments in tracer quantities (1.75 nmol/ml), labeled hydrogen sulfide was produced, and a roughly equal amount of label was incorporated into insoluble material. Methane and carbon dioxide were produced from [methyl-¹⁴C]methionine. Evidence is given favoring methane thiol as an intermediate in the formation of methane, carbon dioxide, and hydrogen sulfide from the terminal methiol group of methionine. Methionine may be an important source of sulfide in lake sediments.     

Purification of the human serum inter-alpha-trypsin inhibitor by zinc chelate and hydrophobic interaction chromatographies

Human inter-α-trypsin inhibitor (ITI) was isolated from a pool of freshly collected human sera by chromatographic steps including DEAE ion-exchange-, zinc-chelate-, and hydrophobic-interaction chromatographies, followed by a final gel filtration. The present results agree with previously reported affinity of ITI for zinc and furthermore provide evidence for the particularly low hydrophobicity of this protein. In each purification step, a control of the chromatographic parameters by fused rocket immunoelectrophoresis led to a standardized mild elution system. The highly purified inhibitor was depleted of any fragments and displayed a fully retained trypsin-inhibiting activity.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

Purification of three γ-chains with different molecular weights from normal human plasma fibrinogen

Three forms of the normal human plasma fibrinogen γ-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique γ-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Aα, Bβ and smallest γ-chain (γ₅₀) eluted at progressively higher ionic strengths, but the elution positions of Aα, Bβ and γ₅₀ chains were identifcal for fibrinogen from each of the three different chromatographic fractions. The unique γ chain of fibrinogen in the second chromatographic peak (γ₅₅) eluted at an ionic strength higher than that of the γ₅₀ chain, while the largest γ-chain (γ_57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the γ-chains unique to them, suggesting that the γ-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three γ-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

Isolation and identification of 5-methylthioribose from Escherichia coli B

5-Methylthioribose was isolated after incubation of Escherichia coli B in a glucose-salts medium. At least 60% of the radioactivity in absolute ethanol extracts of the residue from lyophilized medium supplemented with ³⁵SO₄²⁻ was located in two chromatographic areas that were identified as 5-methylthioribose and its sulfoxide. The sulfoxide was formed by oxidation of 5-methylthioribose during necessary processing of cultures and fractions. These compounds were characterized by functional group analysis and chromatographic comparison with authentic material. 5-Methylthioribose sulfoxide was isolated from 12 l of incubation medium of E. coli. After purification in three paper and one thin-layer chromatographic systems, 50 μg was obtained. The trimethylsilyl derivative of this compound was compared with that of authentic 5-methylthioribose sulfoxide. Gas chromatography and mass spectrometry confirmed the identity. This is the first report of 5-methylthioribose from a bacterium.