scholarly journals A CYTOCHEMICAL STUDY OF CYTOPLASMIC BASIC PROTEINS IN THE ASCIDIAN OOCYTE

1965 ◽  
Vol 25 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Richard Davenport ◽  
Janice C. Davenport
Keyword(s):  
Positive Charge ◽  
Amino Groups ◽  
Acid Dyes ◽  
Fast Green ◽  
Cytochemical Study ◽  
Basic Proteins ◽  
Protein Molecules ◽  
Nucleoprotein Complex ◽  
High Concentrations ◽  
Net Positive Charge

The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins.

A Diagonal Paper Electrophoretic Method for the Selective Isolation of Histidyl Peptides

1971 ◽  
Vol 49 (11) ◽  
pp. 1225-1232 ◽  
Author(s):  
W. H. Cruickshank ◽  
T. M. Radhakrishnan ◽  
H. Kaplan
Keyword(s):  
High Voltage ◽  
Positive Charge ◽  
Proteolytic Enzymes ◽  
Paper Electrophoresis ◽  
Selective Isolation ◽  
Amino Groups ◽  
Electrophoretic Method ◽  
Original Direction ◽  
Citraconic Anhydride ◽  
Net Positive Charge

Thiolysis of an imidazolyl-dinitrophenyl-histidyl peptide at either pH 3.5 or 6.5 results in an increase in the net positive charge on the peptide. It is shown that this property can be used to form the basis of a diagonal paper electrophoretic purification of histidyl peptides from proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is digested with pepsin in 10% formic acid and, if necessary, with other proteolytic enzymes. The enzymatic digest is subjected to high-voltage paper electrophoresis at either pH 3.5 or 6.5. A guide strip is removed, thiolyzed with 2-mercaptoethanol, and subjected to electrophoresis at the same pH at right angles to the original direction of electrophoresis. The histidyl peptides are displaced off the diagonal toward the cathode. The off-diagonal peptides are isolated from the original electrophoretogram by thiolysis and electrophoresis using the diagonal electrophoretogram to locate the positions of the dinitrophenyl-histidyl peptides.

Aldehyde gold clusters for molecular labeling

1995 ◽  
Vol 53 ◽  
pp. 858-859
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya
Keyword(s):  
Covalent Bond ◽  
Aldehyde Group ◽  
Gold Clusters ◽  
Side Reactions ◽  
Amino Groups ◽  
Sodium Cyanoborohydride ◽  
Schiff’S Base ◽  
Protein Molecules ◽  
Hydroxysuccinimide Esters ◽  
Primary Amino

Glutaraldehyde is a useful tissue and molecular fixing reagents. The aldehyde moiety reacts mainly with primary amino groups to form a Schiff's base, which is reversible but reasonably stable at pH 7; a stable covalent bond may be formed by reduction with, e.g., sodium cyanoborohydride (Fig. 1). The bifunctional glutaraldehyde, (CHO-(CH2)3-CHO), successfully stabilizes protein molecules due to generally plentiful amines on their surface; bovine serum albumin has 60; 59 lysines + 1 α-amino. With some enzymes, catalytic activity after fixing is preserved; with respect to antigens, glutaraldehyde treatment can compromise their recognition by antibodies in some cases. Complicating the chemistry somewhat are the reported side reactions, where glutaraldehyde reacts with other amino acid side chains, cysteine, histidine, and tyrosine. It has also been reported that glutaraldehyde can polymerize in aqueous solution. Newer crosslinkers have been found that are more specific for the amino group, such as the N-hydroxysuccinimide esters, and are commonly preferred for forming conjugates. However, most of these linkers hydrolyze in solution, so that the activity is lost over several hours, whereas the aldehyde group is stable in solution, and may have an advantage of overall efficiency.

scholarly journals Proteolytic and peroxidatic reactions of commercial horseradish peroxidase with myelin basic protein

1978 ◽  
Vol 169 (3) ◽  
pp. 567-575 ◽  
Author(s):  
Wendy Cammer ◽  
Lesley Z. Bieler ◽  
William T. Norton
Keyword(s):  
Horseradish Peroxidase ◽  
Myelin Basic Protein ◽  
Basic Protein ◽  
Protein A ◽  
Low Molecular Weight ◽  
Basic Proteins ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations ◽  
A Minor

Degradation of myelin basic protein during incubations with high concentrations of horseradish peroxidase has been demonstrated [Johnson & Cammer (1977) J. Histochem. Cytochem.25, 329–336]. Possible mechanisms for the interaction of the basic protein with peroxidase were investigated in the present study. Because the peroxidase samples previously observed to degrade basic protein were mixtures of isoenzymes, commercial preparations of the separated isoenzymes were tested, and all three degraded basic protein, but to various extents. Three other basic proteins, P2 protein from peripheral nerve myelin, lysozyme and cytochrome c, were not degraded by horseradish peroxidase under the same conditions. Inhibitor studies suggested a minor peroxidatic component in the reaction. Therefore the peroxidatic reaction with basic protein was studied by using low concentrations of peroxidase along with H2O2. Horseradish peroxidase plus H2O2 caused the destruction of basic protein, a reaction inhibited by cyanide, azide, ferrocyanide, tyrosine, di-iodotyrosine and catalase. Lactoperoxidase plus H2O2 and myoglobin plus H2O2 were also effective in destroying the myelin basic protein. Low concentrations of horseradish peroxidase plus H2O2 were not active against other basic proteins, but did destroy casein and fibrinogen. Although high concentrations of peroxidase alone degraded basic protein to low-molecular-weight products, suggesting the operation of a proteolytic enzyme contaminant in the absence of H2O2, incubations with catalytic concentrations of peroxidase in the presence of H2O2 converted basic protein into products with high molecular weights. Our data suggest a mechanism for the latter, peroxidatic, reaction where polymers would form by linking the tyrosine side chains in basic-protein molecules. These data show that the myelin basic protein is unusually susceptible to peroxidatic reactions.

scholarly journals The ‘Chromatoid Body’ in Spermatogenesis

1961 ◽  
Vol s3-102 (58) ◽  
pp. 273-292
Author(s):  
BHUPINDER N. SUD
Keyword(s):  
Phase Change ◽  
Cytoplasmic Inclusion ◽  
Primary Spermatocyte ◽  
Residual Body ◽  
Chromatoid Body ◽  
Acid Dyes ◽  
Basic Proteins ◽  
Daughter Cells ◽  
Final Maturation ◽  
First Time

The chromatoid body was discovered by von Brunn (1876) in the cytoplasm of the young spermatid in the white rat. It was first described in a marsupial by KorfT (1902), in a vertebrate other than mammals by the Schreiners (1905, 1908), and in an invertebrate by Bösenberg (1905). The word chromatoide was first used in connexion with spermatogenesis by Benda (1891), who called this cytoplasmic inclusion der chromatoide Nebenkörper. The German authors generally call it der chromatoide Körper, the French authors corps chromatoïde. Wilson (1913) referred to it as the chromatoid body and it is generally given this name in papers written in English, though the expression ‘chromatic body’ is sometimes used. It is suggested that the ‘residual body’ described by Gresson and Zlotnik (1945) is identical with the chromatoid body of other authors. In most species the chromatoid body is spherical or ovoid but in some it assumes other forms as well and in a few it is never spherical or ovoid. The chromatoid body is usually single in each cell, but sometimes there are 2 or 3 and in a few there are many. In living cell the chromatoid body generally gives a low phase-change, and is invisible or almost invisible when studied by direct microscopy. In the Mammalia, however, it gives a higher phase-change. The chromatoid body is highly resistant to acetic acid. It is deeply stained by basic dyes and basic dye-lakes. It is also stained intensely by acid dyes. The chromatoid body cannot in most cases be blackened by silver or long osmication techniques. The histochemical reactions show that the chromatoid body consists mainly of RNA and basic proteins rich in arginine. There is little or no tyrosine. Lipid, carbohydrates, DNA, alkaline phosphatase, and calcium are not shown by histochemical techniques. As a rule the chromatoid body is homogeneous but in some cases it has a cortex and a medulla. In many cases it is surrounded by a clear, vacuole-like space. Under the electron microscope it has been seen as an opaque irregular body, as an irregular mass of closely aggregated, dense, osmiophil granules, or as a faintly electron-opaque body. The chromatoid body has so far been recorded in certain species of mammals, a bird, reptiles, cyclostomes, Crustacea, insects, and arachnids. In most cases it appears for the first time during the growth of the primary spermatocyte. Its presence in the spermatid has been recorded in practically all cases. With a few exceptions it has not been found to take any obvious part in the final make-up of the spermatozoon. The chromatoid body in most cases seems to disappear at the metaphases of meiosis and to be later reconstructed in the daughter cells. The chromatoid body probably originates from the ground cytoplasm. On the basis of histochemical studies it is tentatively suggested that the function of the chromatoid body may be to provide basic proteins for the final maturation of the chromatin in the nucleus of late spermatids. Certain authors have considered that a cytoplasmic inclusion occurring in the young (and in some cases mature) spermatozooids of certain liverworts, mosses, and a gymnosperm is to be regarded as the homologue of the chromatoid body. Reasons are given for denying this supposed homology.

scholarly journals The mechanism of spray electrification: the waterfall effect

2016 ◽  
Author(s):  
James K. Beattie
Keyword(s):  
Negative Charge ◽  
Positive Charge ◽  
Hydrophobic Hydration ◽  
General Phenomenon ◽  
Aqueous Interfaces ◽  
Preferential Adsorption ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Low Permittivity ◽  
Net Positive Charge

Abstract. The waterfall effect describes the separation of charge by splashing at the base of a waterfall. Smaller drops that have a net negative charge are created, while larger drops and/or the bulk maintain overall charge neutrality with a net positive charge. Since it was first described by Lenard (1892) the effect has been confirmed many times, but a molecular explanation has not been available. Application of our fluctuation-correlation model of hydrophobic hydration accounts for the negative charge observed at aqueous interfaces with low permittivity materials. The negative surface charge observed in the waterfall effect is created by the preferential adsorption of hydroxide ions generated from the autolysis of water. On splashing, shear forces generate small negative drops from the surface, leaving a positive charge on the remaining large fragment. The waterfall effect is a manifestation of the general phenomenon of the negative charge at the interface between water and hydrophobic surfaces that is created by the preferential adsorption of hydroxide ions.

THE EFFECT OF PROCOAGULANT PHOSPHOLIPID VESICLES WITH NET POSITIVE CHARGE ON THE ACTIVITY OF PROTHROMBINASE

1987 ◽  
Author(s):  
J Rosing ◽  
H Speijer ◽  
J W P Govers-Riemslag ◽  
R F A Zwaal
Keyword(s):  
Vitamin K ◽  
Negative Charge ◽  
Positive Charge ◽  
Coagulation Factor ◽  
Phospholipid Vesicles ◽  
Electrostatic Model ◽  
Acidic Phospholipid ◽  
Positively Charged ◽  
Net Positive Charge ◽  
Prothrombin Activation

It is generally thought that procoagulant phospholipid surfaces that promote the activation of vitamin K-dependent coagulation factors should have a net negative charge in order to promote calcium-dependent binding of the enzymes (FVIIa, FIXa and FXa) and substrates (prothrombin and FX) of the coagulation factor-activating complexes. Two models have been proposed to explain calcium-mediated association of vitamin K-dependent proteins with phospholipid: a) an electrostatic model, in which a positively-charged protein-calcium complex is attracted by a negatively-charged phospholipid surface and b) a chelation model in which a coordination complex is formed between calcium ions, γ-carboxyglutamic acids of the proteins and negatively-charged membrane phospholipids. To study the effect of the electrostatic potential of phospholipid vesicles on their activity in the pro-thrombinase complex the net charge of vesicles was varied by introduction of varying amounts of positively-charged stearylamine in the membrane surface. Introduction of 0-15 mole% stearylamine in phospholipid vesicles that contained 5 mole% phosphatidylseri-ne (PS) hardly affected their activity in prothrombin activation. Electrophoretic analysis showed that vesicles with > 5 mole% stearylamine had a net positive charge. The procoagulant activity of vesicles that contained phosphatidic acid, phosphatidylglyce-rol, phosphatidylinositol or phosphatidyl-glactate (PLac) as acidic phospholipid was much more effected by incorporation of stearylamine. Amounts of stearylamine that compensated the negative charge of acidic phospholipid caused considerable inhibition of the activity of the latter vesicles in prothrombin activation. The comparison of vesicles containing PS and PLac as acidic phospholipid is of special interest. PS and PLac only differ by the presence of NH+ 3-group in the serine moiety of PS. Thus, in spite of the fact that vesicles with PLac are more negatively charged than vesicles with PS, they are less procoagulant. Our results show that a) although procoagulant membranes have to contain acidic phospholipids there is no requirement for a net negative charge, b) the amino group of phosphatidylserine has an important function in the interaction of procoagulant membranes with vitamin K-dependent proteins and c) the chelation model can satisfactorily explain calcium-mediated lipid-protein association.

Bimodal Effect of Amphiphilic Biocide Concentrations on Fluidity of Lipid Membranes

1996 ◽  
Vol 51 (11-12) ◽  
pp. 853-858 ◽  
Author(s):  
Marian Podolak ◽  
Dariusz Man ◽  
Stanislaw Waga ◽  
Stanislaw Przestalski
Keyword(s):  
Binding Energy ◽  
Lipid Membranes ◽  
Positive Charge ◽  
Egg Yolk ◽  
Biologically Active ◽  
Double Positive ◽  
Dipole System ◽  
Monovalent Ions ◽  
Single Positive ◽  
High Concentrations

Abstract Using the spin label method (ESR) it has been shown that biologically active, amphiphilic compounds (quaternary ammonium salts -AS) containing polar heads with single and double positive charge caused, at low concentrations, decrease fluidity of liposome membranes formed with egg yolk lecithin (EYL). At higher concentrations an increase in fluidity was observed. With compounds having a single positive charge minimum fluidity of membrane structure occurs in the range of 1 to 3%, with compounds containing double positive charge -in the range of 4 -6 % . That effect does not depend on polar head size and length of alkyl chains of the AS used. Analysis of the electrostatic interaction between positive charges and dipole system suggest that at low ion concentrations the binding energy of the system increases, while it decreases at high concentrations. For the model presented, maxi­mum of binding energy of the system occurs at 3% of positive monovalent ions and at 6% of positive divalent ions admixed.

scholarly journals Chemical reactivity of the functional groups of insulin. Concentration-dependence studies

1984 ◽  
Vol 217 (1) ◽  
pp. 135-143 ◽  
Author(s):  
H Kaplan ◽  
M A Hefford ◽  
A M L Chan ◽  
G Oda
Keyword(s):  
Functional Groups ◽  
Chemical Reactivity ◽  
Pyrex Glass ◽  
Amino Groups ◽  
High Dilution ◽  
No Adsorption ◽  
Adsorption Effects ◽  
Labelling Procedure ◽  
High Concentrations ◽  
Reaction Vessels

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 × 10(-3)-1 × 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 × 10(-3)-1 × 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 × 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 × 10(-5) and 1 × 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 × 10(-3) to 5 × 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 × 10(-6) to 1 × 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.

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