Protease A from cotton seeds. Isolation and purification of the enzyme

Protease A has been isolated in the homogeneous state from dormant seeds of cotton plants of the Tashkent-I variety. A scheme is proposed for the isolation and purification of the enzyme which includes the following stages: extraction of the defatted seeds with 0.1 M phosphate buffer, pH 7.4; precipitation of the protein with ammonium sulfate at 60% saturation; desalting by dialysis; and ionexchange chromatography on a column containing CM- and DEAE-celluloses. The molecular weight of the enzyme has been determined as 60,000. The enzyme efficiently hydrolyzes azocasein and the 7S and 11S reserve proteins of cotton seeds. Its maximum activity appears at pH 6.4–7.4 and a temperature of 35–40°C; it is not activated by sulfhydryl reagents and loses its activity in the presence of diisopropyl phosphorofluoridate. The assumption is made that protease A belongs to the serine type of trypsin-like proteases.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 738–741, November–December, 1986.     

Physicochemical properties and substrate specificity of protease C from dormant cotton seeds

The substrate specificity of a proteolytic enzyme — protease C — isolated from cotton seeds has been studied. The activity of protease C is suppressed completely under the action of diisopropyl phosphorofluoridate. Protein inhibitors — duck ovomucoid, soybean inhibitor, and also TPCK — suppressed the activity of protease C to different degrees. On the basis of results obtained in the hydrolysis of the cottonseed reserve proteins, 7S and 11S globulins, and the B chain of insulin, protease C has been assigned to the serine group of endopeptidases. The optimum conditions — pH, time, and temperature — at which protease C exhibits its maximum activity has been determined.Institute of Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 744–746, September–October, 1988.     

Isolation of a protein with diaphorase activity from cotton seeds and a study of some of its properties

The diaphorase activities of the proteins of different varieties of two species of cotton plant —Gossypium hirsutum L. andG. barbadense L. — have been studied. It has been shown that the proteins of the seeds of the cotton plantG. barbadense possess a low diaphorase activity in comparison with the proteins of the seeds of aG. hirsutum plant. On an electrophoretogram of the proteins, diaphorase activity was localized in two zones, with R_f 0.45 and 0.70. The diaphorase with R_f 0.45 has been isolated by electrophoresis in polyacrylamide gel (PAAG) and some of its properties have been studied. The diaphorase isolated oxidizes NADH and NADPH in the presence of various artificial electron Acceptors, and has two pH optima (at 7.20 and 8.70) and is characterized by relative thermal stability (at 80°C). In the case of the total extract, brief boiling does not lead to inactivation of the enzyme, which shows the presence in cotton seeds of a factor stabilizing this diaphorase. The molecular weight of the proteins isolated, according to gel filtration on Sephadex G-150, is 59,000, and from the results of SDS-PAAG electrophoresis it is 13,600, which shows a tetrameric structure of the enzyme.Institute of Experimental Plant Biology, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 416–421, May–June, 1987.     

Protein kinase from cotton seeds

A Ca²⁺-dependent protein kinase C, active at pH 6.5–8.0 has been found in cotton seeds for the first time. The localization of the enzyme in the seeds has been established and some of its properties are described (stability in various media, capacity for performing the phosphorylation of various substrates, activation by calcium ions). Highly active preparations of cottonseed protein kinase C have been isolated by biospecific chromatography.Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 255–259, March–April, 1989.     

Isolation of protease B from cotton seeds

Protease B has been isolated from dormant cotton seeds by fractionation with ammonium sulfate, ion-exchange chromatography on CM-cellulose, and gel filtration through Acrilex P-10 and Sephadex G-75, with 128-fold purification. The enzyme exists in dimeric and monomeric forms. According to the results of gel filtration, their molecular weights are 72,000 and 36,000, respectively. The enzyme consists of a single polypeptide chain including sugars. The N-terminal amino acid of protease B is alanine. The enzyme possesses proteolytic activity in the pH range from 4 to 6.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 506–510, July–August, 1984.     

Isolation of protease B from cotton seeds

Protease B has been isolated from dormant cotton seeds by fractionation with ammonium sulfate, ion-exchange chromatography on CM-cellulose, and gel filtration through Acrilex P-10 and Sephadex G-75, with 128-fold purification. The enzyme exists in dimeric and monomeric forms. According to the results of gel filtration, their molecular weights are 72,000 and 36,000, respectively. The enzyme consists of a single polypeptide chain including sugars. The N-terminal amino acid of protease B is alanine. The enzyme possesses proteolytic activity in the pH range from 4 to 6.     

Change in the activity of the proteolytic enzymes of cotton seeds when the plants are affected by wilt

The change in the proteolytic activity of the seeds of the cotton plant varieties Tashkent-1- Tashkent-6, S-4727, and 108-F when the plants are affected by wilt has been studied. A relationship has been established between the decrease in activity for seeds of the Tashkent-1 and Tashkent-6 varieties and the degree of attack by wilt. A hypothesis has been put forward on the existence of variety differences in the response reaction of the cotton plant to the introduction of the pathogen. The appearance of distinctive electrophoretic protein components in the seeds of wilt-affected cotton plants of the Tashkent-1 and Tashkent-6 varieties has been shown. A hypothesis has been made of a possible increase in the amount of proteinase inhibitors in response to the infection of the plants by wilt, a consequence of which is a sharp decrease in proteolytic activity and the appearance of distinctive electrophoretic components.     

Change in the activity of the proteolytic enzymes of cotton seeds when the plants are affected by wilt

The change in the proteolytic activity of the seeds of the cotton plant varieties Tashkent-1- Tashkent-6, S-4727, and 108-F when the plants are affected by wilt has been studied. A relationship has been established between the decrease in activity for seeds of the Tashkent-1 and Tashkent-6 varieties and the degree of attack by wilt. A hypothesis has been put forward on the existence of variety differences in the response reaction of the cotton plant to the introduction of the pathogen. The appearance of distinctive electrophoretic protein components in the seeds of wilt-affected cotton plants of the Tashkent-1 and Tashkent-6 varieties has been shown. A hypothesis has been made of a possible increase in the amount of proteinase inhibitors in response to the infection of the plants by wilt, a consequence of which is a sharp decrease in proteolytic activity and the appearance of distinctive electrophoretic components.Institute of Chemistry of Plant Substances of the Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 704–708, September–October, 1987.     

Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus colocynthis

A low molecular weight serine protease from seeds of Citrullus colocynthis was purified to electrophoretic homogeneity with high level of catalytic efficiency (22,945 M^?1 S^?1). The enzyme was a monomer with molecular mass of 25 kDa estimated by SDS–PAGE. The enzyme was highly active over a pH range of 6.5–9.0 and temperature range of 20–80 °C, with maximum activity at pH 7.5 and at 50 °C. The K_m and K_cat were 73 μg/mL and 67/s, respectively. The enzyme was strongly inhibited by PMSF, moderately by soybean trypsin inhibitor, indicating that the enzyme was a serine protease. The enzyme retained 86 and 73% of its activity in the presence of urea and DTT, respectively, and its activity was slightly enhanced in the presence of anionic detergent (SDS). Thus, the enzyme is a novel SDS-stable protease with high catalytic efficiency over wide ranges of pH and temperature which is commercially promising for various industrial applications.     

The change in the globulins during the development of cotton seeds

The biosynthesis of the 11S and 7S globulins — the main reserve proteins of cotton seeds — has been investigated. The periods at which the globulins appear in cotton seeds have been established. The changes in the amino acid composition and in the secondary structure of the 11S globulin during the ripening of cotton seeds have been studied.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 349–355, May–June, 1984.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Purification of angiotensin‐converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity

In the present study, one‐step purification of angiotensin‐converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860‐fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35–40°C and pH 7.4–7.5, respectively. The purity of ACE was determined by SDS–PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC‐MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1–6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half‐maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver–Burk graph.     

Conformational changes of gossypulin in solutions at various pH values influence of salts

Work has continued on the study of the conformational transitions of gossypulin from the seeds of the cotton plant under various conditions. The denaturation of gossypulin as a function of the pH of the medium and the influence of salts (sodium chloride and sodium phytate) on the denaturation process have been studied with the aid of circular dichroism. The gossypulin from cotton plant seeds undergoes complex conformational changes in the pH interval from 2 to 13. Sodium phytate stabilizes the protein molecule at pH 2 and 3.Institute of the Chemistry of Plant Substances of the Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 255–258, March–April, 1987.     

Lipolytic enzymes of cotton seeds V. Triacetinase

Chemistry of Natural Compounds - A triacetinase has been isolated from cotton seeds by the methods of gel filtration and ion-exchange chromatography. The homogeneity of the enzyme was shown by...     

Detection of Adrenaline in Blood Plasma as Biomarker for Adrenal Venous Sampling

An amperometric bi‐enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi‐enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone‐dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.     

Immunochemical characterization of a proteolytic enzyme — Protease A from cotton seeds

It has been established by double immunodiffusion in agar that protease A (a proteolytic enzyme from dormant cotton seeds hydrolyzing the native reserve proteins) is present for the first 3–4 days during the germination of the seeds. An immunological affinity between trypsin and protease A has been revealed which indicates the presence of common structural elements in them.Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 109–110, January–February, 1991.     

Cotton-plant protein isolates

The seeds of the cotton plant are an important source of food protein. The presence of gossypol in the seeds limits the use of protein isolates as additives to food products. The use of salt and alkaline solutions for extracting the proteins leads to the formation of isolates with relatively high gossypol constants. Extraction in an acid medium permits the presence of the toxin to be excluded. In this process, a number of problems arise which are connected with the presence of a large amount of phytin (about 5%) in the seeds of the cotton plant. The latter affects both the yield of food protein on extraction and functional properties. A method is proposed for eliminating traces of phytin from an acid isolate. A substantial influence of phytin on the properties of the proteins has been observed in those cases where the latter is strongly bound to the proteins at acid pH values.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 366–369, May–June, 1983.     

Isolation,purification, and further characterization of an L-phenylalanine oxidase fromMorganella morganii

A l-amino acid oxidase was isolated, purified, and characterized fromMorganella morganii 53187, a bacterium formerly known asProteus morganii. The synthesis of the enzyme by this bacterial strain was growth-associated and decreased sharply when the culture just reached the stationary phase. Based on this finding, the preparation of spheroplast by lysozyme-ethylene-diaminetetra-acetic acid (EDTA) disruption was carried out using the cells harvested during the exponential growth phase. Among several detergents tested, at the detergent-to-protein ratio of 2.5, 3-[(3-cholamidopropyl)dimethylammonio]-l-propane-sulfonate (CHAPS) was very effective in solubilizing most of the enzyme attached to the membranes while still preserving the activity of the solubilized enzyme. The resulting enzyme solution was then purified by hydrophobic interaction chromatography, followed by ion exchange chromatography and gel permeation. The enzyme was purified 19-fold with an overall recovery yield of 12%, corresponding to a specific activity of 252.2 U/mg protein. The selectivity of the purified enzyme toward l-amino acids was pH-dependent. At pH 6.35, the enzyme was very specific to l-leucine, whereas the selectivity for l-phenylalanine could be improved at pH 7.4. The enzyme exhibited a wide optimum temperature range 35–43?C and exhibited 1, l?dimethylferricinium reductase capability in the presence of l-phenylalanine.     

A Functional Role for Aβ in Metal Homeostasis? N‐Truncation and High‐Affinity Copper Binding

Accumulation of the β‐amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer′s disease (AD). The Aβ1–x (x=16/28/40/42) peptides have been the primary focus of Cu^II binding studies for more than 15 years; however, the N‐truncated Aβ4–42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N‐terminal FRH sequence. Proteins with His at the third position are known to bind Cu^II avidly, with conditional log K values at pH 7.4 in the range of 11.0–14.6, which is much higher than that determined for Aβ1–x peptides. By using Aβ4–16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu^II with a conditional K_d value of 3×10⁻¹⁴ M at pH 7.4, and that both Aβ4–16 and Aβ4–42 possess negligible redox activity. Combined with the predominance of Aβ4–42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.     

A Functional Role for Aβ in Metal Homeostasis? N‐Truncation and High‐Affinity Copper Binding

Accumulation of the β‐amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer′s disease (AD). The Aβ1–x (x=16/28/40/42) peptides have been the primary focus of Cu^II binding studies for more than 15 years; however, the N‐truncated Aβ4–42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N‐terminal FRH sequence. Proteins with His at the third position are known to bind Cu^II avidly, with conditional log K values at pH 7.4 in the range of 11.0–14.6, which is much higher than that determined for Aβ1–x peptides. By using Aβ4–16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu^II with a conditional K_d value of 3×10^?14 M at pH 7.4, and that both Aβ4–16 and Aβ4–42 possess negligible redox activity. Combined with the predominance of Aβ4–42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.