Increased Heat Stability Research Articles

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G.

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.

Ovalbumin and angiotensinogen lack serpin S–R conformational change

Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.

Vaccine delivery management.

During the typical 12- to 18-month voyage of a vaccine from manufacturer to immunization site, many situations arise in which the cold chain may be interrupted. Extensive efforts have been made in the 1980s to ensure an uninterrupted cold chain through the use of improved equipment and better training of personnel. One important advance is the vaccine cold-chain monitor, which identifies weak spots in the cold chain and prevents the use of heat-damaged vaccine. Further improvements will require efforts by the recipient countries (e.g., better use of the private sector for transport and equipment management), by donor agencies (e.g., greater consideration of the operational and maintenance costs of the equipment selected and resolution of fuel shortages), and by industry (e.g., more appropriate packaging and pricing of vaccine, extension of the expiration period, and increased heat stability.

Mother-infant face to face interaction: Developmental modifications in sequential properties of the interaction

Heat hardening of wheat (Triticum aesticum L.) resulted in increased heat stability in leaf extracts of fraction I protein, malate dehydrogenase and peroxidase. Polyacrylamide disk gel electrophoretic atterns of fraction 1 protein, malate dehydrogenase and peroxidase did not change after the heat hardening. The individual isozymes of malate dehydrogenase and peroxidase were found to possess the different heat stabilities. After heat hardening only some of the isozymes had an increased heat stability.

Combustibility, smoke evolution, and thermal stability of modified oksalon fibres

A connection has been found between the bromine content of a polymer and its thermal stability, combustibility, and smoke evolution of oksalon fibres. At a bromine content of 4–5% by wt., a sharp increase in heat stability and smoke evolution is observed, plus a decrease in combustibility, which is caused by the formation of network structures.

Electrophoresis and Chromatography of Heat‐Treated Plain, Sugared and Salted Whole Egg

ABSTRACTThe effects of heat treatments on the proteins in plain, 10% sugared and 10% salted whole egg were studied by polyacrylamide disc gel electrophoresis and diethylaminoethyl‐Sephacel ion exchange chromatography. Electrophoretograms of products heated from 57–87°C for 3.5 min showed a wide range of protein stability. Livetins and some globulins were most heat sensitive, while conalbumin and ovalbumin were most stable. Sugar and salt increased heat stability of proteins by 1.6 and 7.9°C, respectively. Heat sensitive proteins were most stabilized by sugar and salt. Heating whole egg and sugared whole egg changed the chromatograms substantially, while heating salted whole egg caused fewer changes.

Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung

NADPH-dependent aldehyde reductase activity in the cytosol of guinea pig lung can be separated into two enzyme species by gel filtration on Sephadex G-100. The major enzyme, purified to homogeneity, has an apparent M r of 86,000 and is composed of single subunits with M r of 23,000. The isoelectric point of this enzyme is 9.8. The enzyme reduces not only aliphatic and aromatic aldehydes and ketones but also quinones. The low reverse reaction is detected only with cyclohexanol as substrate but not with the other alcohols. Although the enzyme utilizes both NADPH and NADH as cofactor, NADPH-dependent activity shows lower K m and V values for substrates than the NADH-dependent activity and the optimal pH is 4.8. The enzyme has higher affinity for NADPH than NADH and is inhibited more strongly by NADP + than by NAD +. The addition of NADPH or NADP + increases heat stability of the enzyme, but NADH and NAD + have no effect. The enzyme is uniquely inhibited by pyrazole as well as quercitrin, benzoate, SH-reagents, and reducing agents e.g., Fe 2+, 2-mercaptoethanol, and dithiothreitol. Pyrazole is a noncompetitive inhibitor with K i values of 84 and 50 μ m with respect to both substrate and cofactor, respectively. The enzyme is electrophoretically and immunochemically different from the minor enzyme with low molecular weight, aldehyde reductase, purified from the same source. Thus, the enzyme is a novel pyrazole-sensitive carbonyl reductase.

Electrophoretic patterns and thermostability of some proteins from heat-hardened wheat

Heat hardening of wheat ( Triticum aesticum L.) resulted in increased heat stability in leaf extracts of fraction I protein, malate dehydrogenase and peroxidase. Polyacrylamide disk gel electrophoretic atterns of fraction 1 protein, malate dehydrogenase and peroxidase did not change after the heat hardening. The individual isozymes of malate dehydrogenase and peroxidase were found to possess the different heat stabilities. After heat hardening only some of the isozymes had an increased heat stability.

Biosoluble polymers for drug delivery

AbstractThe immobilization of α‐chymotrypsin on periodate oxidized cellulose as a biocompatible carrier was investigated. The properties and release of the enzyme from the polymer were studied. It was found that after an initial spurt, the amount of the released enzyme attains a practically constant value and the released enzyme retains its activity and shows increased heat stability. Such systems may be useful for a slow delivery of drugs and enzymes in the body.

Effect of concentration by ultrafiltration on the heat stability of skim-milk

SUMMARYAn examination has been made of the heat stability characteristics of skim-milk concentrate prepared by ultrafiltration (UF). Concentrate prepared by UF was found to be more stable than that prepared by conventional evaporation. In contrast to conventional concentrate, the heat stability of UF concentrate was not appreciably affected by forewarming or addition of permitted stabilizers, but the effect of addition of urea was generally the same for both UF and conventional concentrates; an increase in heat stability was obtained if the milk total solids level was less than 14%. As with conventional concentrate, addition of simple aldehydes induced large increases in the heat stability of UF concentrate. It is suggested that a novel range of sterile milk products could be prepared from UF concentrates. Because of the high protein and low lactose contents of these concentrates, the products might be nutritionally more attractive than those prepared from conventional concentrates.

The synergic effect of urea and aldehydes on the heat stability of concentrated skim-milk

SUMMARYWhen urea is added to concentrated skim-milk (22·5% total solids) no increase in heat stability is observed. However, a similar addition of urea to concentrate stabilized with aldehyde brought about a significant further increase in heat stability. Urea can further stabilize concentrated skim-milk only when aldehyde addition has brought about a change in coagulation mechanism from the normal 2-stage process to a single-stage coagulation. These results can be related to the 2 coagulation mechanisms observed in unconcentrated milks.

Heat stability in concentrated and non-concentrated milks--the effect of urea and beta-lactoglobulin levels and the influence of preheating.

Heat stability as a function of pH has been studied in concentrated and non-concentrated milk systems. The influence of preheating has also been examined. The concentration of beta-lactoglobulin has been shown to affect markedly the heat stability behaviour in both systems but with different characteristics. Increasing the level of urea resulted in increased heat stability in non-concentrated milks, but corresponding concentrated milks showed a small decrease in maximum heat stability. It has not been possible to extrapolate heat stability data from non-concentrated to concentrated milks.

The Synergetic Effect of Urea and Aldehydes on the Heat Stability of Concentrated Skim Milk

When urea is added to concentrated skim-milk (22·5% total solids) no increase in heat stability is observed. However, a similar addition of urea to concentrate stabilized with aldehyde brought about a significant further increase in heat stability. Urea can further stabilize concentrated skim-milk only when aldehyde addition has brought about a change in coagulation mechanism from the normal 2-stage process to a single-stage coagulation. These results can be related to the 2 coagulation mechanisms observed in unconcentrated milks.

Interaction of plasticized polyvinylchloride with oligoester acrylates

By studying the modification of PVC plastisols with oligoester acrylates (OEA) during combined heat treatment new structures were isolated and identified. Using both physical and chemical methods the composition and properties of these structures were determined. It was shown that they are characterized by constant elementary composition, a given degree of crystallinity, increased heat stability and insolubility in known organic solvents. It may be assumed that the structures studied are formed by the interaction between PVC chains and the OEA network.

Effect of urea on the heat coagulation of the caseinate complex in skim-milk

SummaryAdditions of urea progressively increased the heat stability of milk outside of its coagulation time (CT)–pH minimum. In the region of the CT–pH minimum larger amounts of urea were required before an increase in heat stability occurred. The effect of urea was observed over the temperature range 125–140 °Cfornaturalmilk, milk which had been dialysed against synthetic sera, and milk to which a sulphydrylblocking agent had been added. Urea additions did not affect the activation energy of the heat coagulation reaction or the stability of milk to rennet or ethanol.

Altered regulation of cycylic AMP-dependent protein kinase in a mouse lymphoma cell line

The ability of cyclic AMP to inhibit growth, cause cytolysis and induce synthesis of cyclic AMP-phosphodiesterase in S49.1 mouse lymphoma cells is deficient in cells selected on the basis of their resistance to killing by 2 mM dibutyryl cyclic AMP. The properties of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in the cyclic AMP-sensitive (S) and cyclic AMP-resistant (R) lymphoma cells were comparatively studied. The cyclic AMP-dependent protein kinase activity of R cells cytosol exhibits an apparent K a for activation by cyclic AMP 100-fold greater than that of the enzyme from the parental S cells. The free regulatory and catalytic subunits from both S and R kinase are thermolabile, when associated in the holoenzyme the two subunits are more stable to heat inactivation in R kinase than in S kinase. The increased heat stability of R kinase is observed however only for the enzyme in which the catalytic and cyclic AMP-binding activities are expressed at high cyclic AMP concentrations (10 −5—10 −4 M), the activities expressed at low cyclic AMP concentrations (10 −9—10 −6 M) being thermolabile. The regulatory subunit of S kinase can be stabilized against heat inactivation by cyclic AMP binding both at 2 · 10 −7 and 10 −5 M cyclic AMP concentrations. In contrast, the regulatory subunit-cyclic AMP complex from R kinase is stable to heat inactivation only when formed in the presence of high cyclic AMP concentrations (10 −5 M). The findings indicate that the transition from a cyclic AMP-sensitive to a cyclic AMP-resistant lymphoma cell phenotype is related to a structural alteration in the regulatory subunit of the cyclic AMP-dependent protein kinase which has affected the protein's affinity for cyclic AMP and its interaction with the catalytic subunit.

Struktur und Eigenschaften komplexer Gele aus Gelatine und Pektin

The authors studied the effects of aging time and composition on the thermomechanical properties of complex gels of gelatin and pectin which had been produced on the basis of complexes obtained at pH values below the isoelectric point of gelatin. With aging for up to 7 days, the elasticity of the gels decreases and the melting points are raised by 2--4degreesC. An increase in the relative content of pectin in the complex leads to a considerable rise of the melting point of the complex gel. The increase in heat stability of these gels is explained by the development of an ion lattice, which is due to the interaction of the oppositely charged groups of the pectin and the gelatin. Furthermore, hydrophobic interactions between the non-polar groups of the components might be involved.

Characterization of residual enzyme activity in fibroblasts from patients with adenosine deaminase deficiency and combined immunodeficiency: evidence for a mutant enzyme.

A proportion of patients suffering from the autosomal recessive form of severe combined immunodeficiency have an inherited deficiency of adenosine deaminase (EC 3.5.4.4; adenosine aminohydrolase) (erythrocyte isoenzyme). We have, however, found residual adenosine deaminase activity in fibroblasts derived from four such patients. The enzyme responsible for this activity is biochemically homologous with the high-molecular-weight tissue isoenzyme of adenosine deaminase found in normal fibroblasts and tissues other than erythrocytes. The residual adenosine deaminase has an altered electrophoretic mobility, increased heat stability as compared to normals, and can be detected in fibroblasts of obligate heterozygotes. Our previous studies have indicated that the tissue and erythrocyte adenosine deaminase isoenzymes contain a common catalytic unit controlled by the gene affected in severe combined immunodeficiency with absent adenosine deaminase (erythrocyte isoenzyme). This residual adenosine deaminase therefore represents, most likely, a "mutant" enzyme in fibroblasts of patients with severe combined immunodeficiency. The data support the hypothesis that, in these patients, severe combined immunodeficiency is due to a mutation at the adenosine deaminase locus.

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ( v v ) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.

Some properties of alkaline-phosphatases in parenchymal and biliary tract cells separated from rat liver

Livers of both normal and bile-duct ligated rats have been separated into parenchymal cells and fractions enriched in biliary tract cells. The specific activity of alkaline phosphatase increases in both types of cells after occlusion of the bile duct but the increase is more marked in the case of parenchymal cells. The enzyme from parenchymal cells migrates as a single zone on electrophoresis, whereas additional zones of lower anodal mobility occur in extracts of biliary tract preparations. The alkaline phosphatase activity of the slower zones is significantly less stable to heat than that of the zone characteristic of parenchymal cells. It is postulated that the increase in heat stability of alkaline phosphatase in extracts of whole rat liver after bile-duct ligation is due to an increase in the proportion of the more stable form of the enzyme predominant in parenchymal cells.