A versatile gel casting cum electrophoresis apparatus

A simple apparatus for vertical.,in situ, polyacrylamide or agarose gel casting as well as for the subsequent electrophoresis is described. The apparatus is completely leakproof and does not require any special device like clamps, O-rings, gaskets, grease etc. for sealing. Slab gels of various thickness (0.04 to 1.0 cm) can be made and the apparatus can be used for analytical or preparative purposes. Gel rods can also be cast and run in the device. Forward as well as reverse polarity electrophoresis of a sample can be run simultaneously in the apparatus. NCL Communication No.: 3077.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.     

Structure and properties of pectin gels in plant cell walls

Abstract This review deals with recent advances in the structural characterization of pectins and the gels which they form, in relation to auxin-induced extension growth, the ripening of fruit, and cellular recognition. Pectins are block polysaccharides. Heavily branched, largely methyl-esterified blocks alternate with unbranched blocks of varying degrees of esterification. The unbranched, non-esterified blocks can aggregate through calcium binding to form the junction zones that hold a gel together. The aggregates are of two, or possibly four, chains at low calcium levels, and larger with excess calcium. The fall in wall pH during auxin-induced growth activates glycanase enzymes. These may attack some components of the pectic fraction, as well as xyloglucans. Pectin-bound calcium ions may be displaced but this probably has little effect on gel strength. Pectins may be cross-linked by diferulate esters when growth stops. The softening of ripe fruit is due to loss of cohesion in the pectin gel. In apples this results from replacement of the pectins by more esterified forms. In many other fruits it results from depolymerization by polygalacturonases, assisted by pectinesterases, so that the remaining segments are too short for effective calcium binding. Pectins have a further role in the recognition reactions between plant cells and some of their bacterial and fungal pathogens.     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

A highly sensitive fluorometric assay for "enkephalinase," a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins

A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG), was synthesized as a selective substrate for the neutral metalloendopeptidase (EC 3.4.24.11) involved in enkephalin metabolism. This enzyme, designated "enkephalinase," cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG (V = 0.65 mumol/mg protein/min and Km = 45 microM) leading to a fluorescence increase related to the disappearance of intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This change was used for quantitative measurements of "enkephalinase" activity in different tissues and determination of inhibitory potency of various compounds. The substrate is not cleaved by aminopeptidase or dipeptidylaminopeptidase activities and the assay itself is rapid, convenient, and sensitive.     

Calibration of prestained protein molecular weight standards for use in the "Western" or enzyme-linked immunoelectrotransfer blot techniques

Prestained protein molecular weight standards allow easy, direct visual location of electrophoretically transblotted lanes on nitrocellulose. They also provide a simple and accurate means for calibrating the molecular weights of resolved bands. Commercial prestained protein molecular weight standards, however, appear to have significantly different molecular weights from the original unstained proteins. We describe a calibration of these prestained molecular weight standards.     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.     

A biologically active thrombin cleavage product of human serum spreading factor

Purified human serum spreading factor preparations consisting of two immunologically-related, biologically-active proteins of molecular weights approximately 65,000 and 75,000 were incubated with purified hydrolytic enzymes: papain, neuraminidase and thrombin. Biologically active products of the enzymatic digestions were obtained in each case. Digestion of serum spreading factor preparations with thrombin produced a single active form of molecular weight approximately 57,000. Generation of a single molecular weight form of serum spreading factor by thrombin cleavage of the two higher molecular weight forms should simplify studies of the biochemistry and biology of this protein, and may represent a reaction of physiological significance.