Characterization of the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes

A heat-stable polypeptide from rabbit reticulocytes has been previously shown to be required for ATP-dependent protein breakdown (Ciechanover, A., Hod, Y., and Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105) and to form covalent conjugates with proteins in an ATP-requiring reaction (Ciechanover, A., Heller, H., Elias, S., Haas, A.L., and Hershko, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1365-1368). We now describe its purification, characterization, and tissue distribution. Its presence in erythrocytes at high level makes this a possible preferred source in the future.     

A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes

The degradation of denatured globin in reticulocyte lysates is markedly stimulated by ATP. This system has now been resolved into two components, designated fractions I and II, in the order of their elution from DEAE-cellulose. Fraction II has a neutral protease activity but is stimulated only slightly by ATP, whereas fraction I has no proteolytic activity but restores ATP-dependent proteolysis when combined with fraction II. The active principle of fraction I is remarkably heat-stable, but it is non-dialysable, precipitable with ammonium sulfate and it is destroyed by treatment with proteolytic enzymes. In gel filtration on Sephadex-G-75, it behaves as a single component with a molecular weight of approximately 9,000.     

A protein kinase inhibitor in brown adipose tissue of developing rats

A heat-stable protein was extracted from brown adipose tissue of infant rats that inhibited both purified bovine heart protein kinase and a crude preparation of protein kinase from brown fat. It did not act as an adenosine triphosphatase nor did it exert its effect by proteolytic action. Inhibition of protein kinase was affected in both the presence and the absence of cyclic AMP. Most of the inhibitory activity was present in the 100000g supernatant fraction of tissue homogenates. Inhibitory activity was highest perinatally and it declined 10 days after birth. It is suggested that the protein kinase inhibitor may play a role in regulating cyclic AMP actions.     

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosqualatus venom

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10.The anticoagulant principle possesses phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity.The anticoagulant action of the purified principle was competitively inhibited by platelet phospholipid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoaugulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the activation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.     

Participation of the tryptophan synthase inactivating system from yeast in the activation of chitin synthase

The previously described tryptophan synthase “inactivase II”, a proteolytic enzyme from yeast, exhibits high activity in the activation of chitin synthase. Tryptophan synthase inactivase I shows essentially no activity.The purified, heat-stable inhibitor of the tryptophan synthase inactivating enzymes also inhibits the activation of chitin synthase. We take these results to mean that the proteolytic inactivation of tryptophan synthase and the proteolytic activation of chitin synthase are catalyzed and regulated by the same protease/inhibitor system     

An extracellular blood-anticoagulant glycopeptide produced exclusively during vegetative growth by Myxococcus xanthus and other myxobacteria is not co-regulated with other extracellular macromolecules

We have shown that the blood anticoagulant activity (BAA) secreted by Myxococcus xanthus (designated myxaline) is a heat-stable molecule; a high-molecular-mass extracellular fraction also with an apparent BAA is a thermolabile protease. This property allowed us to assay the BAA content in crude boiled culture supernatants and to study the conditions under which it is produced. Heat-stable BAA is strictly extracellular and its production is restricted to vegetative growth in M. xanthus. Unlike the other extracellular proteins, its production is not affected by mutations that regulate secretion; mutations that modify the extracellular proteolytic activity do not modulate the amount of myxaline produced either. Several other species of Myxococcus and one other myxobacterial species produce a heat-stable BAA during vegetative growth.     

Autocrine stimulation of WI38 cell proliferation in the presence of glucocorticoids. Characteristics of the stimulatory factor(s) involved in this response

Chronic exposure to hydrocortisone (HC) or dexamethasone (DEX) results in a 20-40% extension in the proliferative lifespan of WI38 cells. Within a single growth cycle, the addition of HC or DEX at seeding results in saturation densities 20-40% higher than in control cultures. We have recently reported that, within a single growth cycle, the proliferative response of WI38 cells to glucocorticoids is mediated by a stimulatory factor(s) present in medium conditioned by cells in the presence of the hormone. We report here that chronic exposure to medium conditioned in the presence of HC for the first 24 h after seeding (24-h HC-conditioned medium (24-h HC-CM)) results in a 25% extension in the proliferative lifespan of these cultures. The generation of the stimulatory factor(s) present in glucocorticoid-conditioned medium is apparently dependent upon undefined cellular alterations which result from the subcultivation-procedure; confluent or low-density quiescent cultures did not generate media stimulatory to cell growth in the presence of glucocorticoids. This response was not trypsin-dependent, since cultures subcultivated in the absence of proteolytic treatment generated media equally stimulatory to cell growth. A further characterization of this glucocorticoid-induced activity revealed the stimulatory factor(s) was of low MW (dialyzable and recoverable in the less than 10,000 MW fraction following ultrafiltration), heat-stable (95 degrees C), and resistant to treatment with trypsin, chymotrypsin, or protease (S. griseus).     

Purification and Characterization of the Heat-Stable Factors Essential for the Conversion of Lignoceric Acid to Cerebronic Acid and Glutamic Acid: Identification of N-Acetyl-l-Aspartic Acid

Abstract: The conversion of lignoceric acid to cerebronic acid, ceramides, cerebrosides, and glutamic acid is catalyzed by a rat brain particulate preparation. The heat-stable factor, prepared from calf cerebellum, together with the heat-labile factor, a pyridine nucleotide, and Mg²⁺ are essential to all of these metabolic pathways. Our previous work showed that the heat-stable factor is composed of at least two components, HSF-1 and HSF-2, and identified HSF-2 as d -glucose-6-phosphate. In the current investigation, HSF-1 was further purified and found to be N -acetyl- l -aspartic acid. In addition, it was discovered that a third component, HSF-3, is also required for heat-stable factor activity. A reconstituted system composed of N -acetylaspartic acid, glucose-6-phosphate, and HSF-3 fully replaced the heat-stable factor essential for the conversion of lignoceric acid to cerebronic acid and glutamic acid. The reconstituted heat-stable factor did not show the initial time lag always observed with the crude heat-stable factor.     

α-Hydroxylation and oxidation of lignoceric acid in brain: The role of heat-stable and heat-labile factors

Our previous investigations disclosed that the heat-stable and heat-labile factors obtained from brain cytosol are required for -hydroxylation and oxidation of lignoceric acid by rat brain particulate fraction. The heat-stable factor was recently found to contain glucose-6-phosphate, N-acetylaspartate, glutamate, aspartate, glutamine, inorganic phosphate and low levels of adenosine nucleotide as active components. A combination of these compounds was as effective as the crude heat-stable factor for enzymic activity. Using these compounds, we reinvestigated the requirement for the heat-labile factor. With crude heat-stable factor there was an absolute requirement for the heat-labile factor; however, with various combinations of the individual components of the heat-stable factor, some degree of activity was obtained without the heat-labile factor. When aspartate or one of its derivatives, N-acetylaspartate or oxaloacetate, was used in place of the heat-stable factor, the activity was relatively low but highly stimulated by the addition of heat-labile factor. On the other hand, higher activity was obtained when glutamate or one of its derivatives, glutamine or -ketoglutarate, was used without heat-labile factor. The addition of heat-labile factor to this system did not stimulate the activity. When studying the aspartate family, we discovered that the requirement for the heat-labile factor varied in a descending order: N-acetylaspartate > aspartate > oxaloacetate. Lignoceric acid oxidation was further characterized with rat brain particulate fraction, NADPH, Mg²⁺, glutamate, inorganic phosphate, and AMP without heat-stable and heat-labile factors. It was found that the requirement for NADPH was also partially eliminated with glutamate but not aspartate. The effects of various inhibitors, such as inhibitors of the electron transfer system, oxidative phosphorylation, the enzymes involved in citric acid cycle, and glycolysis, suggest that the heat-stable factor is involved in producing ATP or other high energy compounds to be used for the activation of lignoceric acid. ATP added to the system in place of heat-stable factor resulted in less than one-half of the lignoceric acid oxidation.     

Native and latent forms of liver phosphorylase phosphatase. The non-identity of native phosphorylase phosphatase and synthase phosphatase.

The directly measurable (native) phosphorylase phosphatase present in a fresh mouse liver extract is bound to particulate glycogen and is not inhibited by heat-stable inhibitors. Treatment of the extract with trypsin or ethanol at room temperature caused a more than 10-fold increase in phosphorylase phosphatase activity. This increased activity stems from the activation of completely inactive (latent) enzyme, the major part of which is present in the high-speed supernatant. The trypsin-revealed activity can be completely blocked by heat-stable inhibitors. Treatment of the animal with glucocorticoids increases, and fasting decreases the activity of the native phosphorylase phosphatase. The level of latent enzyme, however, is unaffected by these treatments. The major portion of synthase phosphatase in the fresh liver extract is bound to glycogen. This enzyme is inhibited by the heat-stable inhibitor-2 and inactivated by trypsin or ethanol as well as by several treatments that have little effect on phosphorylase phosphatase. Upon DEAE-cellulose chromatography at 0 degrees C of a fresh liver extract, phosphorylase phosphatase and synthase phosphatase were resolved as separate, single peaks. If the preparation was not kept at 0 degrees C during the entire procedure, two peaks of each enzyme were observed. Under these conditions the first peak of phosphorylase phosphatase and of synthase phosphatase coincided. From these findings it is concluded that synthase phosphatase and phosphorylase phosphatase, in their native form, are distinct enzymes.     

Increased ATP-dependent proteolytic activity in lon-deficient Escherichia coli strains lacking the DnaK protein.

Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated protease. Comparison of the levels of ATP-dependent proteolytic activity present in lon rpoH dnaK mutants and in lon rpoH dnaK+ mutants showed that the level of ATP-dependent proteolytic activity was elevated in the lon rpoH dnaK mutant strain. These findings suggest that DnaK negatively regulates a new ATP-dependent proteolytic activity, independently of sigma 32. Other results indicate that an ATP-dependent proteolytic activity was increased in a lon alp strain after heat shock. It is not yet known whether the same protease is associated with the increased ATP-dependent proteolytic activity in the dnaK mutants and in the heat-shocked lon alph strain.     

INHIBITORY EFFECT OF RABBIT SERUM FACTOR ON IN VITRO OOCYTE MATURATION OF THE TELEOST FISH,ORYZIAS LATIPES*

The present study indicates that a factor in rabbit serum inhibits the in vitro steroid- and gonadotropin-induced maturation of oocytes of the teleost fish, Oryzias latipes. Such inhibitory activity could not be recognized in the serum of this fish or in the fluids from mammalian follicles. Passage of the serum inhibitor through a cellulose membrane indicated that it has a molecular weight of less than 3,500. The inhibitory activity on steroid-induced oocyte maturation was not destroyed by heating, by repeated freezing and thawing or by treatment with proteolytic enzymes, lipase or amylases. However, its activity could be removed by extraction with charcoal but not with ethyl ether or toluene. The inhibitory action of the heat-stable and dialyzable serum factor was reversible. The factor appears to exert its inhibitory effect upon the oocyte itself in an early step of maturation, rather than upon the follicle cells.     

The Effect of Growth Regulators and Cu2+ on the Activation of Amylopectin-l,6-glucosidase Activity in Pea Seedlings

No evidence could be obtained for hormonal control of amylopectin-l,6-glucosidase activity in germinating peas for the first 72 hours of germination. The embryonic axis did not stimulate the appearance of enzyme activity. The autolytic system which releases amylopectin-l,6-glucosidase activity from the particulate fraction, in which it originates, was studied in greater detail. Using Cu²⁺ ions to inhibit a proteolytic enzyme in vivo, it was shown that enzyme activation can occur in the zymogen-like granules without liberation of the enzyme into the soluble cell fraction. Activity so formed is labile. Some of the data on proteolytic enzymes in peas is discussed and an attempt made to interpret the liberation of amylopectin-l,6-glucosidase in peas on the basis of the involvement of at least two distinct proteolytic enzymes.     

Repair of radiation-induced damage to the cell division mechanism of Escherichia coli

Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), William D. Fisher, Alice A. Hardigree, and George E. Stapleton. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. Bacteriol. 91:737-742. 1966.-Microscopic observations of irradiated populations of filamentous Escherichia coli cells indicated that filaments can be induced to divide by a substance donated by neighboring cells. We have made this observation the basis for a quantitative technique in which filaments are incubated in the presence of nongrowing donor cells. The presence of "donor" organisms promotes division and subsequent colony formation in filaments. "Donor" bacteria do not affect nonfilamentous cells. An extract of "donor" cells retains the division-promoting activity. The extract has been partially fractionated, and consists of a heat-stable and a heat-labile component. The heat-stable component is inactive in promoting cell division, but enhances the activity of the heat-labile component. The division-promoting system is discussed as a radiation repair mechanism and as a normal component of the cell division system in E. coli.     

Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

A total of 38 lactic acid bacteria, belonging to Lactobacillus, isolated from 24 samples of traditional Egyptian dairy products, were screened for antimicrobial activity against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacterial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable antimicrobial compound showing no reduction in activity after heat treatment from 60 to 100°C for 15 and 30 min. Since it was inactivated by proteolytic enzymes, it is considered to be proteinaceous in nature and, therefore, referred to as a bacteriocin-like substance. This compound was also active over a wide pH range (pH 2–6). The antimicrobial compound was partially purified by 40% ammonium sulfate precipitation. Lactobacillus brevis was tested for its in vitro antibiotics susceptibility, tolerance to bile salts, resistance to low pH values, acidifying activity, proteolytic activity, and haemolytic activity. The results showed the potential of L. brevis strain as a probiotic culture, and hence it can be utilized in the manufacturing of pharmaceuticals and dietary supplements.     

Casein-hydrolyzing activity of sIgA antibodies from human milk

During pregnancy and immediately after delivery (i.e. at the beginning of lactation), the female organism is frequently characterized by an immune status similar to that of patients with autoimmune diseases. In addition, lactation is associated with an appearance of catalytically active antibodies or abzymes (Abzs) with DNAse, RNase, ATPase, amylolitic, protein kinase and lipid kinase activities in breast milk. However, until now there were no examples of human milk Abzs with a proteolytic activity. We present the first evidence that electrophoretically and immunologically homogeneous human milk sIgAs possess a beta-casein-hydrolyzing activity different from known proteases. Abzs specifically hydrolyze both human and bovine beta-caseins but not many other proteins tested. Using different methods including in situ analysis of proteolytic activity in a gel after SDS-PAGE it was shown that the observed proteolytic activity is an intrinsic property of human milk polyclonal sIgAs. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of Abzs, while a specific inhibitor of serine proteases (AEBSF) significantly inhibited the proteolytic activity of the abzymes. The K(M) value for human casein as a substrate was estimated (7.3 microM). Our findings suggest that the immune system of clinically healthy mothers can generate IgAs with a beta-casein-specific serine protease-like activity.     

Characterization of an arginine-specific protein kinase tightly bound to rat liver DNA

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit. The main characteristic of this protein kinase is that it is arginine-specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid-labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP-independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparin have no effect on its activity.     

H2O2 destruction by ascorbate-dependent systems from chloroplasts

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3′-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated.System A is membrane-bound, reacts with 3,3′-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide.The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide.Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H₂O₂ which is produced in these organelles by photosynthetic O₂ reduction.