Myelin basic protein in frozen and unfrozen bovine brain: a study of autolytic changes in situ.

Abstract— Frozen and unfrozen bovine brains were used to determine the extent of in situ degradation of myelin basic protein. The following three parameters were investigated. (1) The time interval between death and sampling of the tissue, (2) the effective temperature of the tissue during this interval, and (3) the effect of freezing and thawing on the subsequent autolysis of myelin basic protein. Polyacrylamide gel electrophoresis was carried out on unfrozen white matter solubilized with phenol-formic acid–water. The resulting electrophoretic pattern showed no qualitative changes in the myelin basic protein after tissue incubation at 4° or 23°C for up to 24 h. When myelin basic protein was extracted, purified and quantitated, there was no apparent decrease within 24 h of incubation at 23°C. However, if the tissue was frozen and thawed prior to incubation, there was a rapid disappearance of myelin basic protein such that only 10% remained after 24 h of incubation. Basic protein extracted from frozen or unfrozen tissue that had undergone autolysis for up to 24 h was found to be encephalitogenic in guinea pigs. Electron microscopy of frozen and thawed material showed separation and fraying of myelin lamellae. It is postulated that the above morphological changes probably render the basic protein readily accessible to proteolytic enzymes.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

Loss of acrosin from bovine spermatozoa following cold shock: Protective effects of seminal plasma

Loss of the alkaline proteinase acrosin and other proteins from the acrosome of bovine spermatozoa was investigated following cold shock and/or incubation of the spermatozoa at either 5, 21, or 37 °C for 4 hr. As detected by electrophoretic analyses of the acrosomal material two bands of acrosin activity and 10 proteins were lost from the acrosome after cold shock and incubation for 4 hr at 5 or 21 °C, whereas one acrosin band and 10 protein bands were lost after cold shock and incubation at 37 °C. Only 45% of the total acrosin activity remained in the acrosome after both cold shock and 4-hr incubation at 37 °C. Egg yolk, present at levels above 15%, and seminal plasma prevented much of the loss of acrosin from the cells.     

Glial fibrillary acidic protein from bovin and rat brain Degradation in tissues and homogenates

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol. wt. polypeptide in bovine tissues incubated at 24 °C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000–40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 °C. At pH 8.0 proteolysis of the glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Rapid changes in chick liver acetyl-CoA carboxylase indicative of phosphorylation control

Liver fatty acid synthesis was suppressed 75,95 and 90% within 1, 2 and 4 hrs respectively of depriving chicks of food. Accompanying this rapid drop in lipogenesis was a marked reduction in acetyl-CoA carboxylase activity, i.e., 40 and 75% decrease after 2 and 4 hrs of fasting. Adding 10mM citrate to the crude liver supernatant, or incubating the supernatant at 37°, 30 min increased activity of the briefly fasted birds, but neither method restored carboxylase activity to fed level. Heat and citrate activation were additive and together resulted in an activity comparable to the fed condition. The heat-dependent activation was accelerated by exogenous phosphoprotein phosphatase, and completely blocked by 100 mM NaF. Thus, enhancement of carboxylase activity from liver of briefly fasted chicks appears to be a dephosphorylation process. This is the first report indicating acute changes in chick carboxylase activity may involve a phosphorylation-dephosphorylation mechanism.     

Ultramicroassay for plasma renin concentration in the rat using the antibody-trapping technique

Using the antibody-trapping technique, picogram quantities of angiotensin-I generated during 24 hr of incubation at 37°C were stable and fully protected against peptidases. The method employs purification of angiotensin-I antisera on DEAE-cellulose and purification of renin substrate by affinity chromatography using specific antirenin antibodies in order to remove endogenous renin. The assay was performed in a single tube without a transfer step in a total volume of 30 μl at pH 6,5 with incubation for 24 hr at 37°C. With a normal rat plasma renin concentration of 5 × 10^?4 GU ml^?1, the detection limit was 10 nl or a total of 5 × 10^?9 GU. In the range 20–125 nl, precision was ±10%.     

Effect of meteorological variates on persistence of primary conidia and capilliconidia of Erynia radicans (Zygomycotin: ntomophthorales) under natural conditions

Persistence of conidia of an isolate of Erynia radicans (Syn. Zoophthora radicans) was investigated in relation to the meteorological conditions which occurred during autumn-winter of 1990–91 in the coastal plain in Israel. Capilljconidia shielded from the sun, placed on the abaxial surface of leaves of Plumeria acuminata, persisted for 24 h to at least 120 h. Exposed capilliconidia, placed on the adaxial surface of the same leaves, died within 24 h. Almost all the primary conidia shielded from the sun (placed on the abaxial surface of the same leaves) died within a single day. Conidial viability was expressed in subsequent germination on an agar medium. Capilliconidial persistence was closely related to the daily air temperatures, expressed as cumulative day-degrees. Differences in relative humidity had no substantial effect on capilliconidial mortality. At daytime temperatures of ≤ 20°C, mortality after 24 h incubation was lowest (≤ 34%) and the persistence duration, longest (at least 120 h). Increases in daytime temperature up to 24°C for a few hours increased mortality (37–57% after 24 h incubation) and shortened the persistence duration (72–120 h). Exposure to 24–29°C during daytime greatly increased mortality (65–58% after 24 h) and further shortened the persistence duration (24–48 h). Daytime temperatures of > 29°C were lethal to all capilliconidia within 24 h. Temperature had a profound effect on capilliconidial persistence also under controlled environmental conditions. The significance of capiliiconidial persistence is discussed in relation to activity of the fungus in its natural environment.     

The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

Glutamine synthetase in cultured whole retinas from the embryonic chick: Enhancement of basal and induced activity by 4 °C storage

Storage of whole retinas from the embryonic chick for 24 h at 4 °C resulted in increased basal levels of glutamine synthetase (GS) during subsequent incubation at 37 °C in the absence of cortisol. GS levels in these retinas maintained initially at 4 °C (CS), in many cases, exceeded GS levels in cortisol-induced whole retinas incubated solely at 37 °C. The increase in basal GS activity is seen within 48 h of the transfer of the retinas from 4 to 37 °C. If cortisol (0.001 μg/ml = 2.8 nm or 0.01 μg/ml = 28 nm) is added during the last 24 h of culture to CS retinas subsequently transferred to 37 °C, levels of GS are attained that are higher than those in the corresponding retinas cultured continually at 37 °C. However, the activity ratios (GS specific activity in cortisol-treated retinas/GS specific activity in retinas not exposed to cortisol) are similar for CS retinas and those maintained at 37 °C throughout. Monolayers of retinal cells display similar basal and cortisol-induced levels of GS independent of treatment. Retinal monolayers maintained at 4 °C for 24 h and subsequently incubated at 37 °C do not exhibit increases in either basal or cortisol-induced levels of GS over those in monolayers maintained at 37 °C throughout. The CS-promoted increase in the basal and cortisol-induced GS activity of whole retinas is eliminated by enzymatic dispersion of the retina just prior to 37 °C culture of the cells as monolayers. Both basal and cortisol-induced GS levels in the latter monolayers resemble those in retinal cells kept as monolayers throughout.     

Determination of Amino Acid Sequence in Surfactin,a Crystalline Peptidelipid Surfactant Produced by Bacillus subtilis

Some conditions of autolysis in cultured tobacco cells were examined for temperature, cell culture age and aeration. Cells autolyzed readily at 45°C. Seventy percent of the dry matter, almost 100% of the soluble sugar, 40% of the insoluble sugar and 60% of the total nitrogen in the initial cells were excreted within 5 hr of incubation in water. At lower physiological temperatures, excreted substances were reabsorbed into cells during the early period of incubation under aerobic conditions.

Rapidly growing cells excreted larger amounts of sugar, nitrogen and solid matter than did non-growing cells during autolysis at 30°C.

Plasmolysis was observed in autolyzed cells.

Autolysis was makedly stimulated by anaerobic conditions.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organophosphorus compounds and carbamates as perilysosomal modifiers

Male rats were administered small doses of malathion over different lengths of time, and lysosomes and lysosome-rich suspensions were prepared from the livers of the experimental and control animals. From the assays of enzyme activity it was concluded that under the conditions of the experiments malathion produced no significant effect upon total acid phosphatase, cathepsin and aryl sulphatase activity. No appreciable effect either was found in the enzyme activities of 10% suspensions of liver homogenates and in the unsedimentable fraction.Malathion administration resulted, however, in an increase in free enzyme activity when lysosome-rich suspensions were preincubated at 37° in isotonic sucrose at pH 5 for up to 120 min. Pretreatment of animals with hydrocortisone was not found to prevent the increase of free enzyme activity.In vitro incubation of lysosome-rich suspensions with malathion, malaoxon, paraoxon, EPN, carbaryl and Boots R.D. 12473 in isotonic sucrose at pH 5 and 37° for various time intervals resulted in an increase of free enzyme activity.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Effect of hypothermia (20–25°C) on mitosis in PtK1 cells

PtK₁ cells enter prophase and complete mitosis at 24–25°C but are inhibited from entering prophase at 20–21°C. Cells which have progressed up to midprophase at 24–37°C return to interphase when cooled to 20–21°C, but those in late prophase complete a normal, although prolonged mitosis. If prophase cells which have reverted to interphase at 20–21°C are incubated at 24–37°C they reenter prophase and complete mitosis. This temperature-induced prophase-interphase-prophase transition can be repeated several times on the same cell. At 24–25°C the process of spindle formation (i.e. prometaphase to the initiation of anaphase) encompasses approximately 75% of the total mitotic interval, with a duration of 8–12 h, compared to about 50% of the mitotic interval and a duration of 0.5 to 1.0 h at 37°C.     

Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation

Body wall that mainly consists of collagen and polysaccharides is the edible part of sea cucumber and is easy to go autolysis, while the proteinase(s) responsible for autolysis remains unclear. In the present study, a gelatinolytic metalloproteinase (GMP) from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatographic steps including DEAE-Sephacel, Sephacryl S-200, Q-Sepharose and Phenyl-Sepharose. The molecular mass of GMP as estimated by SDS-PAGE and gelatin zymography was 45 kDa. The enzyme revealed high activity at a slightly alkaline pH range (8.0–9.0) and the optimal temperature was at 40–45 °C. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the activity, whereas other proteinase inhibitors did not show any effect. Peptide mass fingerprinting of the enzyme obtained 3 peptide fragments with a total of 58 amino acid residues, which was 91.4% identical to an alkaline metalloprotease from Pseudomonas fluorescens, strongly suggesting it is a metalloproteinase. Divalent metal ion Ca²⁺ is essential for its activity, indicating it is a calcium-dependent metalloproteinase. Furthermore, GMP hydrolyzed collagen effectively at 37 °C and gradually even at 4 °C, suggesting its involvement in the autolysis of sea cucumber.     

Glutamine synthetase in cultured whole retinas from the embryonic chick

Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37°C is preceded by storage at 4°C for 2–24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37°C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4°C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37°C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4°C storage when compared with whole retina controls incubated at 37°C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4°C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37°C. This suggests that enhancement of GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4°C storage.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Functional enrichment of mannanase-treated spent brewer yeast

In this study, the effect of Bacillus amyloliquefaciens-produced β-mannanase on the nutrient diffusion (release) and antioxidant activity of spent brewer yeast (SBY) was investigated. Three pretreatments were performed: (1) autolysis at 50°C for 24?h; (2) autolysis at 50°C for 24?h, with the addition of β-mannanase during the autolysis; (3) autolysis at 50°C for 24?h, and the β-mannanase was added for another 12?h treatment. The pretreatments with the addition of β-mannanase caused significant cell wall degradation, markedly increased the yield of SBY extracts. More importantly, this study found that polysaccharides were degraded to be oligosaccharides with a considerable reduction in molecular weights. Meanwhile, pretreatment with the enzyme also exhibited a higher antioxidant activity in SBY extract compared to autolysis itself. The current study indicated that pretreatment (3) had a better effect than pretreatment (2) in terms of improving in antioxidant activity in SBY extract. These improved characteristics of SBY extracts isolated through enzymatic treatment appear to show promising features for their prospective use as natural functional agents.