Separation of transfer RNA and 5S ribosomal RNA using capillary electrophoresis

Capillary gel electrophoresis and capillary electrophoresis using entangled polymer solutions was investigated for their applicability for the separation of low-molecular-mass RNAs (transfer RNA and 5S ribosomal RNA), with a size range of 70–135 nucleotides, from bacteria. Cross-linked polyacrylamide gel-filled capillaries (3 and 5%) were used for capillary gel electrophoresis. Good resolution was obtained suing gel-filled capillaries only for small tRNAs with lengths to 79 nucleotides, larger tRNAs and 5S rRNA could not be resolved using this method. Buffers containing sieving additives were employed to improve separations of RNA by capillary electrophoresis using entangled polymer solutions. The use of linear sieving polymers in buffers resolved 5S rRNA and tRNAs, even when they possessed only different secondary structure or small differences in length (1–5 nucleotides).     

Characteristics of the 5S rRNA from cotton seeds

The individuality of the 5S rRNA from cotton seeds has been determined by the production of a single narrow band on gel electrophoresis and by the characteristic UV spectrum with values of the ratios E₂₆₀/E₂₃₀ and E₂₆₀/E₂₈₀ of 2.03–2.1 and 2.08–2.14, respectively. The melting curve of the 5S rRNA has been obtained and a total hyperchromic effect of 25% has been calculated. The nucleotide composition of the 5S rRNA has been established by determining the amount of nucleotides in an alkaline hydrolysate (22.6% of AMP; 30.2% of GMP; 22.6% of UMP; and 24.6% of CMP). The nucleotides were identified from the position of their appearance in the elution profile, from their UV spectra in 0.1 N HC1 and 0.1 KOH, and also by one- and two-dimensional chromatography of the nucleotides obtained, in the presence of markers.     

Characteristics of the 5S rRNA from cotton seeds

The individuality of the 5S rRNA from cotton seeds has been determined by the production of a single narrow band on gel electrophoresis and by the characteristic UV spectrum with values of the ratios E₂₆₀/E₂₃₀ and E₂₆₀/E₂₈₀ of 2.03–2.1 and 2.08–2.14, respectively. The melting curve of the 5S rRNA has been obtained and a total hyperchromic effect of 25% has been calculated. The nucleotide composition of the 5S rRNA has been established by determining the amount of nucleotides in an alkaline hydrolysate (22.6% of AMP; 30.2% of GMP; 22.6% of UMP; and 24.6% of CMP). The nucleotides were identified from the position of their appearance in the elution profile, from their UV spectra in 0.1 N HC1 and 0.1 KOH, and also by one- and two-dimensional chromatography of the nucleotides obtained, in the presence of markers.Institute of the Chemistry of Plant Substances of the Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 817–821, November–December, 1980.     

Isolation of ribosomes from cotton seeds

Summary 1. It has been established that the method of precipitation with ethanol in the presence of an increased concentration of Mg²⁺ can be used for the rapid isolation of the ribosome fraction from plant material.2. The optimum conditions for the complete precipitation of the ribosome fraction from a homogenate of cotton seeds have been selected.3. By treating an extract of the seeds with 0.5 M KCl it was possible to reduce the amount of tRNA in a preparation of the total rRNA.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 852–856, November–December, 1977.     

Isolation of the total tRNA from cotton seeds

Summary 1. A comparative study has been made of various methods of isolating the tRNA from cotton seeds. It has been found that the best results are given by extraction of cotton seeds with a mixture of 80% phenol and 0.14 M NaCl.2. The conditions have been worked out for the preparative isolation of the total tRNA.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 621–624, September–October, 1978.     

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.     

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 immunosimilar proteins from cotton seeds by immunoaffinity chromatograph

A sorbent highly specific forVerticillium proteins has been obtained from BrCN-Sepharose and rabbit immunoglobulins. By affinity chromatography using this sorbent a protein immunologically similar to the proteins of the mycelium of the fungusV. dahliae has been isolated from cotton seeds of the Tashkent-1 variety. The molecular mass of the protein has been determined, and its proteolytic activity has been established.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 746–749, September–October, 1993.     

Isolation of immunosimilar proteins from cotton seeds by immunoaffinity chromatograph

A sorbent highly specific forVerticillium proteins has been obtained from BrCN-Sepharose and rabbit immunoglobulins. By affinity chromatography using this sorbent a protein immunologically similar to the proteins of the mycelium of the fungusV. dahliae has been isolated from cotton seeds of the Tashkent-1 variety. The molecular mass of the protein has been determined, and its proteolytic activity has been established. Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 746–749, September–October, 1993.     

Isolation and characterization of protease inhibitors from cotton seeds

Protease inhibitors of protein nature have been isolated from dormant cotton seeds. The participation of protease inhibitors in the mechanism of protecting the plant from wilt damage is discussed.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 89 14 75. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 445–448, May–June, 1995. Original article submitted June 27, 1994.     

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.     

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.