Major Functional Properties Research Articles

Expression, Characterization, and Crystallization of the Catalytic Core of the Human Insulin Receptor Protein-tyrosine Kinase Domain

The deduced primary sequence of the cytoplasmic protein-tyrosine kinase domain of the insulin receptor contains a conserved kinase homology region (receptor residues 1002-1257) flanked by a juxtamembrane region and a C-terminal tail. A soluble 48-kDa derivative (residues 959-1355) containing these regions but lacking the first six residues of the juxtamembrane region had earlier been synthesized in Sf9 cells using a baculovirus expression system. The catalytic core of the kinase domain was studied first by proteolytic analysis of the 48-kDa kinase and then by expressing a series of truncated kinase domains in transiently transfected COS cells. Based on these studies, two core kinases of 34 (residues 985-1283) and 35 (residues 978-1283) kDa, respectively, were overexpressed in Sf9 cells. Biochemical characterization of the 35-kDa kinase revealed that the core kinase conserved the major functional properties of the native receptor kinase domain. Activity of the 35-kDa kinase toward a synthetic peptide increased more than 200-fold upon autophosphorylation, which occurred exclusively at Tyr-1158, Tyr-1162, and Tyr-1163; the largest increase was observed between bis- and trisphosphorylation of the kinase. The activated 35- and 48-kDa kinases were similar with respect to specific activity and ATP and Mg2+ requirements for peptide phosphorylation. Moreover, autophosphorylation appeared to initiate predominantly at Tyr-1162, immediately followed by phosphorylation at Tyr-1158 and then at Tyr-1163. The rate of autophosphorylation was dependent on enzyme concentration, consistent with a trans-phosphorylation mechanism. Finally, the 35-kDa kinase was crystallized, making possible elucidation of its three-dimensional structure by x-ray crystallography.

Significance of positively charged amino acids for the function of the Acidovorax delafieldii porin Omp34

The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.

Significance of positively charged amino acids for the function of theAcidovorax delafieldiiporin Omp34

The functional significance of charged amino acids of the anion-selective porin Omp34 from Acidovorax delafieldii was investigated by means of conductance measurements. Chemical modification of Lys and Arg as well as titration of charges by adjusting the pH value revealed that positively charged amino acid residues determine the major functional properties of the porin. Positive charges are involved in creating the protein surface potential, the selectivity filter inside the channels, and the voltage-sensing and/or gating mechanisms.

Single-stranded DNA binding protein encoded by the filamentous bacteriophage M13: structural and functional characteristics.

The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation. It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane. The protein is 87 amino acids long and its biological functional entity is a homodimer. In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed. In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed. Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins.

Multiple subpial transection: a new approach to the surgical treatment of focal epilepsy

A new operative approach has been designed for the relief of medically intractable focal epilepsy. It is intended particularly to be used in those cases where the epileptogenic lesion lies in "unresectable" cortex; that is, those cerebral regions subserving speech, memory, and primary motor and sensory function. The procedure is based upon experimental evidence indicating 1) that epileptogenic discharge requires substantial side-to-side or horizontal interaction of cortical neurons, and 2) that the major functional properties of cortical tissue depend upon the vertical fiber connections of the columnar units. The technique requires severing of tangential intracortical fibers while preserving the vertical fiber connections of both incoming and outgoing nerve pathways and of the penetrating blood vessels which also have a vertical orientation. In this study, the effect of multiple subpial transection was assessed on both function and seizure control. The effect on function was reviewed in 32 cases; only 20 cases were evaluated with respect to seizure control, since a follow-up period of 5 years or more (5 to 22 years) is required before conclusions can be drawn. Multiple subpial transection was applied to the precentral gyrus in 16 cases, the postcentral gyrus in six, Broca's area in five, and Wernicke's area in five. With respect to function, the major finding was that none of the 32 patients has suffered a clinically significant behavioral deficit (although subtle deficits could be detected by careful neurological examination). Complete control of seizures has been obtained in 11 (55%) of the 20 cases evaluated. Nine patients developed recurrent seizures consequent to progressive disease unsuspected before operation (Rasmussen's encephalitis in five, tumor in three, and subacute sclerosing panencephalitis in one). In none of these cases, however, did the recurrent seizures arise in the transected zone. Thus, the results indicate that multiple subpial transection is about as effective as standard excisional therapy, and can be successfully employed when epileptogenic lesions encroach upon cortical territories, the removal of which would be functionally incapacitating.

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria

A homogeneous cytochrome P-450 scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregenenolone to be catalyzed by single species of cytochrome P-450 scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450 scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with M r 27 000 and 22 000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, M r of about 14 000. Cytochrome P-450 scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450 scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.

Influenza A hemagglutinin-specific T cell clones strictly restricted by HLA-DR1 or HLA-DR7 molecules.

The antigenic specificities, major histocompatibility complex restrictions and functional properties of influenza virus-specific proliferative cloned cell lines have been studied. These lines were specific for the H3 hemagglutinin subtype of influenza A viruses. By using a large panel of HLA-phenotyped antigen-presenting cells, it was found that the polymorphic structures, defined as DR1 and DR7 molecules, or closely associated structures, function as the restricting elements. We excluded for these lines a possible restricting role of supertypic specificities, known cross-reacting elements on DR molecules, or products of other loci in known linkage disequilibrium with the HLA-DR molecules. Such exquisitely restricted clones might be of great help in the class II typing of antigen-presenting cells. Their specific activity was stable for several months. This has allowed the study of some functional properties of these long-term-cultured cloned cell lines: interleukin 2 sensitivity and production, helper function in specific antibody synthesis and ability to stimulate in mixed leukocyte reactions.

Lecithin production and utilization

AbstractCommercial lecithin is the most important byproduct of the edible oil processing industry because of its functionality and wide application in food systems and industrial utility. The recovery of lecithin from oil is a relatively simple process. Hydration of the phosphatides by water or steam followed by recovery by centrifuge and drying is all that is required. But in order to maximize lecithin's utility and functionality, processing conditions all the way back to the bean or seed must be carefully controlled. Bean storage and handling, crude oil storage, refining pretreats, drying processes, bleaching, chemical modification, and storage all can affect lecithin quality and performance. The effects of processing on lecithin quality and performance is one of the major focal points in this presentation. Utilization of lecithins has expanded beyond the traditional application in paints, chocolate, and margarine. Food technologists have used lecithin as a functional ingredient in many modern systems. Its multifunctional properties and its “natural” status make commercial lecithin an ideal food ingredient. The major functional properties include: emulsification, instantizing and particle wetting, release, viscosity modification and nutrition. The nutritional impact of lecithin is currently being assessed in the medical field as an important factor in improving neurochemical disorders. Other medical and health related activity areas include positive changes in cholesterol, blood chemistry and circulatory factors. Lecithin is also used in numerous industrial and nonfood applications such as pigment dispersing, mold release, and animal feeds. The major source of commercial lecithin is from the processing of soybean oil. Evaluation of lecithins from other seed crops such as cotton, corn, and rapeseed is being pursued. The growth of these sources will be a function of demand.

Functional properties of proteins in foods: A survey

Proteins for foods, in addition to providing nutrition, should also possess specific functional properties that facilitate processing and serve as the basis of product performance. Functional properties of proteins for foods connote the physicochemical properties which govern the behavior of protein in foods. This general article collates the published information concerning the major functional properties of food proteins, e.g., solubility, binding properties, surfactant properties, viscogenic texturizing characteristics, etc. The effects of extraction and processing on functional properties and possible correlations between structure and function are discussed, in relation to practical performance in food systems. Modification of proteins to improve functional characteristics is briefly mentioned.