Low Molecular Weight Constituents Research Articles

Environmental adaptation of proteins: Strategies for the conservation of critical functional and structural traits

1.1. Comparative studies of lactate dehydrogenases (LDHs) and skeletal muscle actins from vertebrates adapted to widely different temperatures and hydrostatic pressures reveal major conservative trends in protein evolution and adaptation. For enzymes, ligand binding, as estimated by apparent Michaelis constant (Km) values, is strongly conserved at physiological temperatures, pressures, intracellular pH values and osmotic compositions of different organisms. The catalytic rate constants (kcatvalues) of enzyme homologues are highest for enzymes of low-body-temperature organisms, a trend that can be interpreted in terms of temperature compensation of metabolism. For skeletal muscle actins. the enthalpy and entropy changes accompanying the assembly of filamentous (F) actin from globular (G) actin are highest in high-body-temperature species and especially low in polar and deep-sea fishes. The thermal stability of G-actin is positively correlated with adaptation temperature, except in the case of actins of deep-sea fishes, which are also highly heat stable. Hydrophobic interactions between actin subunits may be of reduced importance in low-body-temperature animals and, especially, in deep-sea fishes. The differences in enthalpy and entropy changes during the G-to-F transformation favor a close conservation of the equilibrium constant for actin assembly under physiological conditions of temperature and pressure for different species. These adaptive patterns in enzymes and actin are likely to reflect changes in protein primary structure.2.2. The appropriate values for protein traits such as ligand binding abilities and catalytic rates are also shown to be established by the composition of the low molecular weight constituents of the cytosol. For example, the use of a combination of urea and methylamine solutes for osmoregulation by marine elasmobranchs is shown to be a mechanism which permits the conservation of key protein traits at high osmolarities. The methylamine solutes such as trimethylamine-N-oxide have effects on proteins opposite to those of urea, and at the approximately 2:1 concentration ratio of urea to methylamines, these counteracting effects are virtually complete.3.3. Regulation of hydrogen ion activity (pH) also is shown to play a major role in the conservation of critical protein traits. The importance of temperature-dependent pH in ectotherms is discussed in terms of stabilizing binding abilities and maintaining correct regulatory and structural sensitivities of proteins. The buffering capacity of tissues reflects the potential of the tissue for generating acidic end-products during anaerobic metabolism. Skeletal muscle, especially white locomotory muscle of fishes, is highly buffered relative to red locomotory muscle and heart muscle. Much of the difference in buffering may be due to different amounts of histidine-containing dipeptide buffers in the tissues. However, comparisons of muscle-type (M4) and heart-type (H4) LDHs show that the M4 isozyme has approximately twice the number of histidine residues per tetramer of the H4 isozyme. This difference between M4 and H4 isozymes which occur predominantly in anaerobically- and aerobically-poised tissues, respectively, suggests that selection for buffering capacity may be a heretofore unappreciated facet of protein evolution.

Inhibition of FSH action on granulosa cells by low molecular weight components of follicular fluid

Immature female rats were injected with a single dose (10 IU) of pregnant mare serum gonadotropin to induce growth and maturation of ovarian follicles. Using such ovaries as a model, we tested the effects of low molecular weight subfractions of charcoal-adsorbed bovine follicular fluid (FF-c) on (a) radioiodinated human FSH ( 125I-hFSH) binding to ovarian homogenates, (b) ovine FSH-stimulated adenylate cyclase activity in granulosa cell homogenates and (c) cAMP production by intact granulosa cells. The follicular fluid was fractionated by ultrafiltration through membranes of differing pore-sizes into low molecular weight components of 1000–5000 (passing Amicon HIP-5 hollow fibers but retained by Amicon UM-2 membrane) and 500–1000 (passing Amicon UM-2 membrane but retained by Amicon UM-05 membrane). These low molecular weight fractions inhibited 125I-hFSH binding to ovarian receptors, FSH-stimulated cAMP production by rat granulosa cells and FSH-stimulated, as well as fluoride-ion-stimulated adenylate cyclase activity in granulosa cell homogenates. Inhibition of FSH-stimulated adenylate cyclase activity by the FF subfractions was non-competitive as determined by double reciprocal plot analysis. Our results suggest that modulation of FSH effects on granulosa cells may be mediated by low molecular weight constituents of follicular fluid.

Isolation of a low molecular weight constituent from Kentucky blue grass pollen possessing carrier activity of high molecular weight constituent(s).

An immunologically active fraction, designated as C-I-3 was isolated from the dialysate of the aqueous extract of defatted Kentucky Blue Grass pollen by differential dialysis and gel filtration. This fraction was shown to consist of a glycoprotein with a molecular size corresponding to 4,800 daltons and with a pI value of 3.6, and to contain all amino acids except cysteine. The intraperitoneal administration of C-I-3 in saline into mice or rats prior to their immunization with the nondialyzable constituents of the aqueous extract of Kentucky Blue Grass (i.e., the retentate, R) in presence of Al(OH)3 resulted in the enhanced production of anti-R IgE antibodies. The enhancement of the anti-R murine IgE response was shown in adoptive cell transfer experiments to be due to the generation of R-specific T helper cells. Hence, it is suggested that the structural elucidation of C-I-3 may prove useful in establishing the nature of carrier determinant(s) of the major allergenic components of Kentucky Blue Grass pollen.

Studies on Low Molecular Weight Constituents in High Density Polyethylene Containers

Studies on Low Molecular Weight Constituents in High Density Polyethylene Containers

The apoprotein fraction of the bile lipoprotein complex: isolation, partial characterization and phospholipid binding properties

A bile apoprotein fraction (Apo BLC) was isolated by preparative isoelectric focusing (I.E.F.) from the detergent-free form of the bile lipoprotein complex (BLC). Analytical I.E.F. of Apo BLC yields a characteristic and reproducible pattern of two narrow acidic bands (pI 4,8-5,0). This apoprotein presents a strong tendency to undergo self-aggregation in aqueous buffer. A low molecular weight constituent of Apo BLC has been isolated after gel filtration, its mean Mw is estimated by SDS-PAGE at 7,500 daltons. The binding capacity of Apo BLC for phospholipids was investigated on dimyristoylphosphatidylcholine liposomes by gel filtration and zone electrophoresis. The resulting structures, larger than the original single-shelled vesicles, acquire and anodic electrophoretic mobility. Apo BLC has a weaker affinity for lysophosphatidylcholines: these phospholipids decrease the degree of aggregation of the apoprotein. These studies contribute additional data concerning the high affinity of Apo BLC for phosphatidylcholines, which are the major phospholipid constituents of bile. The discussion deals with the fact that association of Apo BLC with bile phosphatidylcholines may present some implications in the pathogeny of LpX and in the process of intestinal fat absorption.

The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.

Autolytic methods for the reduction of the purine content of baker's yeast, a form of single‐cell protein

AbstractWhen a suspension of baker's yeast was incubated at 50°C, the cells became permeable to H+, a process completed in about 4 h. Cellular constituents of low molecular weight then diffused into the medium, but only after a long lag period were cell nucleic acids and proteins degraded by intracellular enzymes. The lag period was shorter at higher temperatures, when RNase and proteinases suffered some thermal denaturation. It was abolished if, before autolysis at 50°C, yeast was plasmolysed by ethyl acetate, or heated to 70°C for 1 min, treatments which, it is thought, disorganised the membrane of the yeast cell vacuole, within which the enzymes are located. Heat treatment, when effective, was sufficiently severe to disorganise the yeast cell plasma‐lemma, with the consequence that a single adjustment of pH immediately following heat treatment served to maintain a constant pH, without addition of buffer, during the subsequent incubation at 50°C. Autolysis was not found to be an entirely suitable procedure for the reduction of the nucleic acid content of baker's yeast, because of the concomitant and severe loss of protein.

A Method for Determining Plasma Proteins in Inflammatory Exudate Absorbed by Sephadex

Dry cross-linked dextran (Sephadex pharmaceutical grade, Debrisan) was applied to weeping sores. After some exudate had been absorbed the gels were removed. The amount of exudate was determined by isotope dilution techniques. The accuracy of the method was tested by determination of plasma proteins in samples of diluted serum containing known concentrations of some plasma proteins. The method seems suitable for determining various high and low molecular weight constituents of exudates.

CHEMORECEPTION IN THE SHRIMP, PALAEMONETES PUGIO: COMPARATIVE STUDY OF STIMULATORY SUBSTANCES IN HUMAN SERUM

1. A bioassay procedure is described for studying a chemically mediated feeding response in the shrimp, Palaemonetes pugio. The procedure involves the attraction of shrimp to a small target in compartmented plastic boxes.2. Bioassays of purified components of human serum showed that the response of shrimp to serum is due to a mixture of substances including both proteins and substances of low molecular weight.3. A dialyzed mixture containing six types of serum proteins accounted for approximately one-fourth of the potency of serum. Although all of the proteins possessed a certain degree of activity, low density lipoprotein and the alphaglobulins were individually the most active components of the mixture.4. A mixture containing the 37 major low molecular weight organic constituents of serum accounted for only approximately one-eighth of the potency of serum. Glycine, taurine and glutamic acid were the most active constituents of this mixture that were tested individually.5. A mixture containing the six types of serum proteins together with the 37 low molecular weight constituents was fully as active as the total serum.6. Ultrafiltration of body fluids or extracts from coquina, crab, lobster, oyster and shrimp showed that in some preparations the major stimulants were large molecules (greater than ca. 1000 MW) whereas in others they were small molecules.

Biochemical and immunological properties of low molecular weight constituents of Timothy pollen antigen.

Biochemical and immunological properties of low molecular weight constituents of Timothy pollen antigen.

Heavy lysozymuria after X-irradiation of the spleen in human chronic myelocytic leukaemia.

BY electrophoretic analysis we have found large quantities of an exceptionally basic protein in the urine of five out of seven consecutive cases of chronic myelocytic leukaemia. In four cases the specific proteinuria occurred when induction of remission was achieved after X-irradiation directed against the enlarged spleen. In one case the protein was found before X-ray therapy, but irradiation evoked an increasing protein response. The proteins have been isolated and characterized as relatively low molecular weight constituents with the enzymatic properties of lysozyme.

The chemistry of furfuryl alcohol resins

AbstractFurfuryl alcohol resins contain a broad spectrum of compounds. The distribution of these compounds in a given resin varies with the manufacturing conditions. A resin containing a high proportion of low molecular weight constituents has been prepared and separated into fractions. Twelve compounds were separated from the resin; four of these are new compounds. The lower molecular weight constituents can be arranged into four major classes: polyfurfuryl alcohols, polyfurfuryl furans, methyl‐substituted polyfurfuryl furans, and polyfurfuryl ethers.

High Temperature Relaxation Transitions in Elastomers

Abstract 1. For binary mixtures of polybutadienes with differing molecular weights, a high temperature (or low frequency) transition was discovered, with the temperature-frequency position of the transition showing up as a function of the molecular weight of the low molecular weight constituents, and yet the transition does not depend on the concentration of these constituents. The value of the maximum for the mechanical loss tangent is determined by the concentration of low molecular weight components. 2. With regard to the viscoelastic behavior of the mixes at reduced temperatures, the transition reflects mainly the presence of the low molecular weight components, but at high temperatures the transition is indicative of the presence of the high molecular weight constituents. 3. The observed transition is associated with the manifestation of mobility of the molecules of the low molecular weight components, as a whole.

A study of furfuryl alcohol resin components by gel permeation chromatography

Furfuryl alcohol resins contain a broad spectrum of compounds. A resin containing a high proportion of low molecular weight constituents has been prepared, and twelved compounds, five of which were previously unreported, have been separated from the resin and characterized. Both qualitative and quantitative gel permeation chromatographic data were obtained for a number of these compounds, and calibration curves have been prepared from the data. The gel permeation chromatographic technique was also used to study the reaction products formed by several of these compounds.

Sephadex Equilibrium-Diffusion Technique for Fractionating Whey and Skimmilk Systems

An equilibrium-diffusion process was developed which utilizes Sephadex G-25 for removing low molecular weight solute materials from whey and skimmilk systems. Two successive treatments by the process removed a total of about 67% of the minerals and about 51% of the lactose from acid whey and about 36% of the minerals and about 72% of the lactose from concentrated skimmilk. Equilibration of the low molecular weight constituents between the milk serum phase and the interior of the Sephadex beads was complete within 1min or less. The extent of fractionation achieved is primarily a function of the mixing ratio of product and Sephadex and the number of successive contact treatments used. The equilibrium-diffusion technique may be useful for developing large-scale, continuous processes for fractionating whey and skimmilk systems into low molecular weight and colloidal constituents.

Introduction to the Proceedings of the Symposium on Urinary Constituents of Low Molecular Weight

IT HAS OFTEN BEEN SAID that all of the concepts necessary for the complete automation of clinical chemistry procedures are already in existence-if we only knew where to look for them! We are now beginning to search for these concepts, as well as to develop new ones that will apply specifically to clinical chemistry. r lie existing analytic technics that are available for routine use in many chemical and biologic laboratories provide one fruitful source. These technics are frequently more accurate, yield a higher resolution, and involve a higher degree of automation than those found in the cliiiical laboratory. A significant effort is now being made to adapt them for clinical research. Other efforts, concerned with new and automated laboratory procedures for tile clinical laboratory, are also being pursued. One of these latter efforts is the Body Fluid Analysis Irogram at the Oak Ridge National Laboratory. This joint program. under the sponsorship of the National Institute of (ieneral Medical Sciences and the U. S. Atomic Energy Commission, is part of the Molecular Anatomy Program (1) at Oak Ridge, in which work is progressmc on the separation and the cataloging of the molecular constituents of human cells and physiologic fluids. Since it is now generally accepted that the majority of human diseases will ultimately be understood at the molecular level, such work should have direct application to the clinical laboratory. The Body Fluid Analysis Program, established after discussions held at the recent conference on Automation in Clinical Laboratories (2), is a multidiscipline effort in which engineers, physical scientists, and life scientists work as a team toward the goal of developing high resolution, automated, analytic systems for body fluids. Such an approach is much like other ellorts at Oak Ridge; that is, many scientific principles and enriueering skills are combined at a single facility to solve complex problems. Since the scope of the iraining and the education of many of the

Use of Nuclear Magnetic Resonance in Determining Molecular Structure of Urinary Constituents of Low Molecular Weight

Abstract Nuclear magnetic resonance is applied to elucidate organic compounds of low molecular weight that occur in urine. Spectrums of specific compounds are shown, as are closely related compounds, to illustrate NMR principles and their application in practice.

Separation of urinary constituents of low molecular weight on Sephadex G 10.

Jagenburg, R., Rödjer, S. & Samsioe, G. Separation of Urinary Constituents of Low Molecular Weight on Sephadex G 10. Scand. J. clin. Lab. Invest. 22, 54-56, 1968. Low molecular urinary constituents have been separated by gel filtration on Sephadex G 10 using distilled water or 0.2 M acetic acid as eluant. The aliphatic amino acids were completely separated from urea, uric acid, creatinine and chlorides but incompletely separated from glucose and phosphates. The separation between the basic amino acids and the other aliphatic amino acids was improved by using 0.2 M acetic acid as eluant. The aromatic amino acids, phenylalanine and tyrosine had larger elution volumes than inorganic salts. Tryptophan was strongly retained by the gel.

The Enzymatic Reduction of Nicotinamide N-Oxide

Abstract An enzymatic system which reduces nicotinamide N-oxide to nicotinamide was isolated from hog liver and purified about 280-fold. The reduction is dependent on the presence of reduced diphosphopyridine nucleotide or a low molecular weight constituent found in boiled liver supernatant. The two cofactors appear to be involved in the expression of two distinct enzymatic activities. Evidence has been presented which indicates both activities may be associated with a single protein or enzymatic unit. The DPNH-dependent reaction is the following. H+ + DPNH + nicotinamide N-oxide → DPN+ + nicotinamide + H2O The nature of the supernatant-dependent reaction is not completely understood, since the identity of the compound which is oxidized in the course of reduction of the N-oxide is not known.

Effects of various soybean products on flatulence in the adult man.

ConclusionsIn 4 human male subjects, kept on a carefully controlled diet to which various fractions of soybean meal were added, the flatus-producing factor in soybeans was concentrated primarily in the low molecular-weight constituents. These experiments also showed that the soybean hulls, fat, water-insoluble polysaccharides, and protein are not associated with flatulence production to any significant degree. Caseinate and soybean proteinate appear to inhibit flatulence. When equivalent amounts of navy bean meal were consumed, flatus volumes increased 2.52 times that following the consumption of dehulled, defatted soybean meal.