Properties of the adenylate cyclase system of isolated hepatocytes following freezing-thawing

The effect of various regimen of freezing and thawing on the functional state of one of the most important receptor-regulatory cell systems, i. e. adenylate cyclase complex (ATP-pyrophosphate lyase cyclating E C 4.6.1.1.), has been studied on isolated rat hepatocytes. Basal and fluoride-stimulating activity and enzyme susceptibility to the regulatory effect of isoproterenol, adrenaline and noradrenaline were shown. Regardless of the regimen used, freezing and thawing decrease the stimulating effect of adrenergetic agents on adenylate cyclase system of isolated hepatocytes. The reason of it is probably the damage of a receptor site. The functional properties of catalytic enzyme subunit practically do not change. The results obtained show the necessity of correction of metabolic cell responses mediated by adenylate cyclase system during their freezing and thawing.     

Characteristics of proteinaceous additives in stabilizing enzymes during freeze-thawing and -drying

Protein-stabilizing characteristics of sixteen proteins during freeze-thawing and freeze-drying were investigated. Five enzymes, each with different instabilities against freezing and dehydration, were employed as the protein to be stabilized. Proteinaceous additives generally resulted in greater enzyme stabilization during freeze-thawing than sugars while the degree of stabilization for basic lysozyme and protamine were inferior to that of neutral and acidic proteins. Freeze-drying-induced inactivation of enzyme was also reduced by the presence of a proteinaceous additive, the extent of which was lower than that for a sugar. In both freeze thawing and freeze drying, the enzymes stabilization by the proteinaceous additive increased with increasing additive concentration. The enhancement of enzyme inactivation caused by pH change was also reduced in the presence of proteinaceous additives. The combined use of a sugar such as sucrose and dextran tended to increase the stabilizing effect of the proteinaceous additive.     

Reversible Photoinhibition in Antarctic Moss during Freezing and Thawing

Tolerance of antarctic moss to freezing and thawing stress was investigated using chlorophyll a fluorescence. Freezing in darkness caused reductions in Fv/Fm (ratio of variable to maximum fluorescence) and Fo (initial fluorescence) that were reversible upon thawing. Reductions in Fv/Fm and Fo during freezing in darkness indicate a reduction in the potential efficiency of photosystem II that may be due to conformational changes in pigment-protein complexes due to desiccation associated with freezing. The absorption of light during freezing further reduced Fv/Fm and Fo but was also reversible. Using dithiothreitol (DTT), which inhibits the formation of the carotenoid zeaxanthin, we found reduced flurorescence quenching during freezing and reduced concentrations of zeaxanthin and antheraxanthin after freezing in DTT-treated moss. Reduced concentrations of zeaxanthin and antheraxanthin in DTT-treated moss were partially associated with reductions in nonphotochemical fluorescence quenching. The reversible photoinhibition observed in antarctic moss during freezing indicates the existence of processes that protect from photoinhibitory damage in environments where freezing temperatures occur in conjunction with high solar radiation levels. These processes may limit the need for repair cycles that require temperatures favorable for enzyme activity.     

Effect of freezing-thawing on the adenylate cyclase activity of the rat liver

Various regimes of freezing and thawing as well as adrenaline and fluoride ions are studied for their effect on the adenylate cyclase activity in liver tissue preparations. The reduction of basal and fluoride-stimulating adenylate cyclase activity and a decrease in the adrenaline-stimulating activity of the enzyme after freezing and thawing are shown. Freezing and thawing are studied for molecular mechanisms of their damaging effect on adenylate cyclase.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

川西亚高山森林林窗不同时期土壤转化酶和脲酶活性的特征

为了解川西亚高山森林林窗对不同时期土壤生态过程的影响,于2012年6月—2013年5月期间,根据温度动态过程,对比研究了生长季节(土壤完全融化期、生长季节前期和生长季节后期)与非生长季节(冻结初期、深冻期和融化期)川西亚高山粗枝云杉(Picea asperata)人工林林窗中心、林缘和林下土壤有机层和矿质土壤层转化酶和脲酶活性变化过程。结果表明:林窗不同区域中,土壤有机层转化酶活性均高于矿质土壤层;在生长季节,土壤有机层和矿质土转化酶活性表现为:林窗中心林下林缘,而脲酶活性表现为:林窗中心林缘林下。冻结初期和深冻期林窗中心土壤转化酶活性均高于林缘和林下,而在融化期林下转化酶活性高于林窗中心和林缘;冻结初期和融化期林下土壤脲酶活性显著高于林窗中心和林缘,而在深冻期林窗不同区域土壤脲酶活性没有显著差异。林窗不同区域在不同时期对土壤转化酶和脲酶活性的响应有着深刻影响。     

Formate dehydrogenase from Clostridium acidiurici

Partial purification of formate dehydrogenase from Clostridium acidiurici has been accomplished, and some properties of the enzyme have been determined. The molecular weight of the protein is at least 200,000 daltons. The enzyme showed marked instability to freezing and thawing and was inhibited strongly by oxygen and by light. Such inhibition was not reversed by incubation in the presence of thiol compounds. Cyanide inhibited the enzyme 90% at 0.1 mm concentrations, but ethylenediaminetetraacetate produced only slight inhibition at concentrations as high as 50 mm. The purified enzyme showed no ferredoxin activity in the Clostridium pasteurianum clastic system during pyruvate oxidation. Crude preparations of the enzyme could be coupled through ferredoxin to the reduction of nicotinamide adenine dinucleotide during formate oxidation, but the purified enzyme could not catalyze the reduction of pyridine nucleotides by formate in the presence of ferredoxin. Formate oxidation with the purified enzyme was readily coupled to benzyl viologen reduction, in which case ferredoxin was not required. An exchange between formate and bicarbonate was catalyzed by both crude and purified preparations of the enzyme, but the net synthesis of formate from CO(2) was not accomplished.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

Enzymic hydrolysis of sphingomyelin by rat liver

1. An enzyme that hydrolyses sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was isolated from rat liver. 2. The enzyme is particle-bound (mitochondria or lysosomes) and can be solubilized by ultrasonic treatment and freezing and thawing. 3. It has been partially purified by precipitation at pH5.2, neutralization and ammonium sulphate fractionation. 4. The enzyme is activated by Triton X-100 (0.2%) or low concentrations of cetyltrimethylammonium bromide (0.02%), higher concentration being inhibitory. 5. The optimum pH is 5-5.5. 6. Of synthetic substrates tested, the erythro isomers of dl-trans-2-N-palmitoyl-1-O-phosphorylcholinesphingosine or dihydrosphingosine were hydrolysed at a rate similar to the natural compound. The threo isomer was hydrolysed much more slowly. The enzyme had little activity on lecithin. 7. The split products of the hydrolysis have little inhibitory effect.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stability Properties of Activated Tryptophan Hydroxylase from Rat Midbrain

Abstract: Time courses of the activation-inactivation sequence in rat midbrain tryptophan hydroxylase after preincubation with calcium, ATP + MgCl₂, or sulfhydryl reagents and after freezing and thawing suggest that the activated enzyme is more vulnerable to loss of activity. The sequence induced by calcium was prevented by the protease inhibitor leupeptin, and an accelerated decline in activity after activation by ATP + MgCl₂ was reduced greatly by increasing levels of tetrahydrobiopterin (BH₄) cofactor. The effects of calcium and ATP + MgCl₂ were additive, which suggests independent mechanisms. The findings suggest that time courses of enzyme activation and inactivation processes may offer a useful way to study the influence of a range of effectors on tryptophan hydroxylase function.     

Reduction of photosynthetic apparatus plays a key role in survival of the microalga Haematococcus pluvialis (Chlorophyceae) at freezing temperatures

The microalga Haematococcus pluvialis is a biotechnologically important microorganism producing a ketocarotenoid astaxanthin. Haematococcus exists either as metabolically active vegetative cells with a high chlorophyll content or astaxanthin-rich haematocysts (aplanospores). This microalga featuring outstanding tolerance to a wide range of adverse conditions is a highly suitable model for studies of freezing tolerance in phototrophs. The retention of H. pluvialis cell viability after freezing–thawing is ascribed to elevated antioxidant enzyme activity and high ketocarotenoid content. However, we report that only haematocysts characterized by a lower photosynthetic activity were resistant to freezing–thawing even without cryoprotectant addition. The key factors of haematocyst freezing tolerance were assumed to be a low water content, rigid cell walls, reduction of the membranous structures, photosynthesis downregulation, and low chlorophyll content. Collectively, viability of Haematoccus after freezing–thawing can be improved by forcing the transition of vegetative cells to freeze-tolerant haematocysts before freezing.

     

α-Crystallin protects human arginosuccinate lyase activity under freeze-thaw conditions

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.     

Injury to human granulocytes at low temperatures

Granulocytes differ from other blood cells in that they are more sensitive to injury on freezing and thawing. Previous studies suggest that the difficulty in preserving them is related to their sensitivity to osmotic stress. A miniaturized system both for freezing granulocytes and testing their function in the same Terasaki plates has been developed. This allowed study of several factors simultaneously including concentration of protective additive, different cooling conditions, and dilution conditions on rewarming.We observed two types of injury to granulocytes frozen to higher subzero temperatures and thawed directly. The first type was initially severe but decreased with time in the frozen state under some conditions and appears not to have been reported in other cell systems. The second type of injury consists of conventional loss of function with longer holding times after freezing. Cells surviving these two classes of injury could be protected against the further stress of rapid cooling into liquid nitrogen, but this protection required a longer time during cooling in the frozen state than with other cell types.We have studied the interactions between several variables, e.g., time in DMSO before freezing and dilution rate after thawing in an attempt to characterize the unusual injurious mechanism at high subzero temperatures that, we believe, is the real cause of the difficulty of preserving these cells.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

Partial purification and characterization of the enzyme which converts precursor liver protein to factor X.

A rat liver post-microsomal supernatant enzyme, which carries out an epigenetic conversion of a protein contained in liver microsomes to Factor X, has been partially purified 250-fold in 50% yield by a combination of salt fractionation and gel filtration. The crude enzyme is stable to freezing and thawing but unstable at 4 degrees C. However, the partially purified enzyme is more stable at 4 degrees C. It requires Ca2+ and HCO3 minus for optimum formation of Factor X activity. The supernatant enzyme is vitamin K dependent and exhibits its maximum rate of formation of Factor X between pH 8 and 8.5.     

Isolation, purification and stability of a glutamin(asparagin)ase preparation from Pseudomonas boreopolis 526

A technique for purification of glutamine asparaginase from Pseudomonas boreopolis 526 is described which provides a 37% yield of the enzyme homogeneous according to electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. The effect of pH, freezing, thawing and lyophilic drying on the stability of glutamine asparaginase was studied. The enzyme is rather stable at pH 4.8 and 4 degrees C. Lyophilic drying of the homogeneous enzyme without addition of stabilizers resulted in a decrease of its activity an is accompanied by formation of protein conglomerates with molecular weights of 280,000 and 660,000 D. Freezing and thawing decreased the activity of the nature enzyme by 40-50%.     

Protein kinase activity at the inner membrane of mammalian mitochondria.

This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.     

Changes in prostaglandin concentration in blood subjected to repetitive freezing and thawing

Successive freezing and thawing of whole blood results in a consistently higher yield of various prostaglandin (PG) compounds. Evaluations were made with radioimmunological assay. The increase in PG concentrations seems to be more associated with cell fragmentation and not with the dissociation of albumin-PG complex. Our data suggest that there may be some dissociation of non-albumin-PG complexes. Artifactually high PG concentrations due to in vitro PG synthetase activity appears minimal at least with respect to indomethacin blocking of this enzyme. There are, in general, only slight differences in PG concentrations in samples with and without indomethacin.