Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa

Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1-3). These comprise 30%, 10%, and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr approximately 150,000) composed of polypeptides ranging in size from Mr 14,000 to 50,000 (alpha 1-alpha 6; 50,000, 38,000, 32,000, 20,000, 16,000 and 14,000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1-A9; Mr 49,000 to 39,000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24,000, 23,000 and 20,000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1/B2). Subunits (Mr approximately 50,000-65,000) are assembled as trimers (8S) or larger oligomers (12S-15S) in mature seeds. The lower molecular weight albumins (LMW1-3) are acidic (pI less than 6), and consist of sets of disulphide-bonded polypeptides (Mr 15,000 and 11,000).     

Phosphorylation of cottonplant proteins under the action of protein kinase inhibitors and activators

The occurrence of Ca²⁺-, Ca²⁺-phospholipid-, Ca²⁺-calmodulin-, and cAMP-dependent phosphorylation has been shown with the aid of protein kinase activators and inhibitors and by electrophoresis and autoradiography. Specific substrates have been revealed and it has been demonstrated that the pathways of the realization of the action of cAMP-dependent and Ca²⁺-calmodulin-dependent protein kinases may intersect.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 627071. Institute of Nuclear Physics, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 442603. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 96–100, January–February, 1994.     

Investigation of the dynamics of the accumulation of some secondary metabolites of cottonplant leaves

The compositions of the secondary metabolites (SMs) of the leaves and petioles of cotton plants of the deciduous and selection lines L-275, L-470, L-475, and 142-F and the variety Tashkent-1 have been studied. It has been shown that the level of -tocopherol and polyprenols in the leaf blades and petioles changes fairly sharply according to the phases of ontogenesis. The results of an exogenous treatment of the leaves of cotton plants of the control varieties Tashkent-1 and 108-F with the SMs showed an interrelationship of deciduousness and the quantitative composition of the sterols.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, Vol. 33, No. 1, pp. 113–117, January–February, 1997.     

Comparative characteristics of proteins of cottonplant lines differing in the strength of the fiber

The enzymes and proteins of the fibers of two lines of cotton plant differing in the strength of the fiber have been investigated. It has been shown that the activities of glucan synthetase and peroxidase rise as the fiber matures, while the activities of β-(1-3)-glucanase and cellulase fall. The specific enzymatic activities of peroxidase and glucan synthetase in the L-175 line, distinguished by a stronger fiber, are higher than for the L-466 line with a weaker fiber. The activity of glucanase changes according to the strength of the fiber. In a study of the protein composition of cotton fibers, polypeptides with molecular masses of 28 and 39 kDa were found among the proteins responsible for the strength of the fiber. A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 530–536, July–August, 1998.     

Preparation of affinity sorbents based on monoclonal antibodies to cottonplant membrane proteins

An immunosorbent has been obtained by conjugating mcABs 2C8.C7 to membrane proteins from cotton seedlings with BrCN-Sepharose 4B. As a result of the affinity chromatography of the total lectin-like proteins of the cotton plant on this sorbent, two fractions of polypeptides have been isolated, and these have been subjected to electrophoretic analysis. A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 807–811, November–December, 1998.     

An investigation of the membranotropicity of the ethylene- and cytokinin-binding proteins from cottonplant shoots by the fluorescent probe method

A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 627071. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 135–136, January–February, 1994.     

Atomic-force-controlled capillary electrophoretic nanoprinting of proteins

The general nanoprinting and nanoinjection of proteins on non-conducting or conducting substrates with a high degree of control both in terms of positional and timing accuracy is an important goal that could impact diverse fields from biotechnology (protein chips) to molecular electronics and from fundamental studies in cell biology to nanophotonics. In this paper, we combine capillary electrophoresis (CE), a separation method with considerable control of protein movement, with the unparalleled positional accuracy of an atomic force microscope (AFM). This combination provides the ability to electrophoretically or electroosmotically correlate the timing of protein migration with AFM control of the protein deposition at a high concentration in defined locations and highly confined volumes estimated to be 2 al. Electrical control of bovine serum albumin printing on standard protein-spotting glass substrates is demonstrated. For this advance, fountain pen nanolithography (FPN) that uses cantilevered glass-tapered capillaries is amended with the placement of electrodes on the nanopipette itself. This results in imposed voltages that are three orders of magnitude less than what is normally used in capillary electrophoresis. The development of atomic-force-controlled capillary electrophoretic printing (ACCEP) has the potential for electrophoretic separation, with high resolution, both in time and in space. The large voltage drop at the tip of the tapered nanopipettes allows for significant increases in concentration of protein in the small printed volumes. All of these attributes combine to suggest that this methodology should have a significant impact in science and technology.      

Distortion of the electrophoretic titration curves of some proteins

The electrophoretic titration curves of complex mixtures of vitamin K-dependent human blood proteins and proteins of Bothrops asper venom were investigated. In both protein mixtures some curves exhibited marked distortions such as additional maxima and minima when Pharmalyte 3-10 carrier ampholytes were used for isoelectric focusing in agarose gels. The distortions result from an unspecific interactions between some carrier ampholyte constituents with particular proteins. The interacting carrier ampholyte components could be completely removed by binding to albumin and ultrafiltration through a UM-2 Amicon membrane with resultant regular titration curves. The interacting carrier ampholyte species were only partially removed by ultrafiltration through a UM-2 membrane without incubation with albumin.     

A structural-functional study of cottonplant glycoproteins

The results are presented of a structural-functional investigation of two groups of cottonplant glycoproteins — lectin-like and extensin-like proteins — using the methods of molecular modeling of various structural fragments of the glycoproteins.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 162 70 71. Translated fromKhimiya Prirodnykh Soedinenii, No. 3, pp. 376–384, May–June, 1999.     

Carbon microspheres with supported silver nanoparticles prepared from pollen grains

In this paper, we present a new method to fabricate carbon microspheres with supported silver nanoparticles on the surfaces. In this method, pollen grains were first treated with AgNO(3) aqueous solution, then preoxidized in air at 300 degrees C and carbonized in nitrogen at 600 degrees C, resulting in the silver/carbon nanocomposites. The silver/carbon nanocomposites were characterized by means of SEM, TEM, TG, and XRD. The size and distribution of the silver nanoparticles on the carbon microsphere surface could be controlled by tuning the AgNO(3) treatment conditions.     

Two-dimensional electrophoretic analysis of human milk proteins

Identification of human milk proteins and formulation of a two-dimensional map is a first step in a project which intends to survey human milk proteins by two-dimensional electrophoresis. Thirty-four proteins have been identified using the Iso-Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species-specific, and, therefore, breast feeding confers a decided advantage for the infant. Using antisera for identification has revealed relationships between molecules which have not been previously noted. The antibody recognizes a common epitope between the IgA alpha chain and lactoferrin, and between the IgD d chain and beta casein. Milk protein concentrations vary longitudinally, diurnally, and individually. Identification of the proteins contributes meaning to the varying patterns. Knowledge of human milk proteins will help to elucidate human nutrition and health needs.     

Flavonoid composition of the pollen (pollen pellets) fromSalix caprea andS. alba

Chemistry of Natural Compounds -     

Comparative analysis of the electrophoretic composition of proteins from native and transformed strains of the mung-beanBradyrhizobium sp.Vigna radiata L.

An experiment on the transfer into the mung-bean rhizobia gene of a recombinant plasmid containing an insect toxin gene (pCaVItoxneo) was performed in order to protect the root nodules from parasitic insects. Transformed strains (MTL) are obtained. The frequency of transformation was 10⁻⁶. A study of the protein composition showed that the transformed strain MTL 12 contained an additional three polypeptides with MM 83, 93, and 98 kDa; the transformed strain MTL 17, one additional polypeptide with MM 83 kDa. Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 651–653, September–October, 1999.     

Cystic fibrosis proteins detected by electrophoretic techniques

For the detection of the cystic fibrosis protein (CFP) in serum of cystic fibrosis (CF) carriers, thin-layer polyacrylamide gel isoelectric focusing proved inappropriate as a diagnostic test, but was useful for screening fractions on purification of CFP by chromatofocusing on a Mono P column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis an Mr 12,000 protein (P12) was found in most CFP-positive sera, indicating good correlation between these two CF-associated proteins. Detection of the P12 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was well reproducible and less delicate than IEF. The technique was also used to purify P12 from serum by two successive preparative electrophoresis steps in a 7.5-15% gradient and 15% homogeneous gel. The use of silver staining revealed that P12, which was present in all sera of CF patients and carriers with variable intensities, was also present in trace amounts in normal sera.     

Schematic structure of a fragment of the cottonplant lignin macromolecule

A schematic structure has been proposed for an average fragment of the macromolecule of cottonplant dioxane lignin on the basis of the results of a chemical study and quantitative¹H and¹³C NMR spectroscopy.Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 234–236, March–April, 1996. Original article submitted June 27, 1994.     

Capillary electrophoretic separations of proteins using carrier ampholytes

Capillary separations of proteins using carrier ampholytes are performed between an anolyte and a catholyte of same pH (pH 3). Depending upon the concentration of carrier ampholytes used, two different separation processes take place. At a 10% concentration, the high-resolution separation of six model proteins is achieved, which can be described as a transient capillary isoelectric focusing (cIEF) system moving isotachophoretically. The isotachophoretic (ITP) behaviour of the system is evidenced by the influence of the catholyte concentration on the separation. The separation is neither pure cIEF nor pure cITP and the migration order of the proteins results from the influence of both their isolelectric points and their mobilities.     

Influence of buffer composition on the capillary electrophoretic separation of carbon nanoparticles

Carbon nanoparticles obtained from the flame of an oil lamp were examined by means of capillary electrophoresis. The influence of buffer composition on the separation of the mixture of negatively charged carbon nanoparticles was studied by varying buffer selection, pH, and concentration. The electrophoretic pattern was affected by both the co- and counter-ion in the buffer solution, influencing selectivity and peak shape. The capillary electrophoretic separations at different pH revealed species with large electrophoretic mobilities under a wide range of pH. The mobility of selected species in the mixture of nanoparticles showed a strong dependence upon the solution ionic strength. The mobility of these nanoparticles as a function of ionic strength was compared to classical electrokinetic theory, suggesting that under the experimental conditions utilized, the species are small, highly charged particles with appreciable zeta potentials, even at low pH.