Crystalline inorganic pyrophosphatase isolated from baker's yeast

Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40 degrees C. and pH 7.0 at a concentration of 3 to 4 x 10(-3)M Na(4)P(2)O(7) and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na(4)P(2)O(7) or K(4)P(2)O(7) proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme.     

Isolation of hexokinase from baker's yeast

1. A method is described for the isolation of hexokinase from baker''s yeast. The method is based mainly on fractionation with alcohol and results See PDF for Structure in a 30-fold increase in specific activity. The final product could be crystallized from ammonium sulfate without change in specific activity. 2. The enzyme catalyzes a transfer of phosphate from adenosinetriphosphate to glucose, fructose, or mannose, the relative rates with these three sugars being 1:1.4:0.3. 3. With glucose as substrate, the turnover number for the crystalline enzyme is 13,000 moles of substrate per 10⁵ gm. of protein per minute at 30° and pH 7.5. The temperature coefficient (Q _10°) between 0 and 30° is 1.9. 4. Magnesium ions are necessary for the activity, the dissociation constant for the Mg⁺⁺ -protein complex being 2.6 x 10^–3. Fluoride in concentrations as high as 0.125 M has no inhibitory effect on the enzyme when the Mg⁺⁺ and orthophosphate concentrations are 6.5 x 10^–3 M and 1 x 10^–3 M, respectively. 5. The crystalline enzyme shows a loss in activity when highly diluted. This loss in activity can be prevented by diluting in the presence of small amounts of other proteins. Of the various protective proteins tested, insulin was the most effective, providing complete protection in a concentration of 6 micrograms per cc.; with serum albumin, a concentration of 60 micrograms per cc. was necessary. Thiol compounds (cysteine, glutathione) exerted no protective action. 6. The inactivation of the crystalline enzyme on incubation with trypsin can be prevented to a marked degree by the presence of glucose. The instability of crude preparations of yeast hexokinase may be attributed to the presence of proteolytic enzymes, since glucose or fructose has a remarkable protective effect on such preparations.     

CRYSTALLINE RIBONUCLEASE

1. A crystalline enzyme capable of digesting yeast nucleic acid has been isolated from fresh beef pancreas. 2. The enzyme called "ribonuclease" is a soluble protein of albumin type. Its molecular weight is about 15,000. Its isoelectric point is in the region of pH 8.0. 3. Ribonuclease splits yeast nucleic acid into fragments small enough to diffuse readily through collodion or cellophane membranes. 4. The split products of digestion, unlike the undigested yeast nucleic acid, are not precipitable with glacial acetic acid or dilute hydrochloric acid. 5. The digestion of yeast nucleic acid is accompanied by a gradual formation of free acid groups without any significant liberation of free phosphoric acid. 6. Ribonuclease is stable over a wide range of pH even when heated for a short time at 100°C. Its maximum stability is in the range of pH 2.0 to 4.5. 7. Denaturation of the protein of ribonuclease by heat or alkali, or digestion of the protein by pepsin, causes a corresponding percentage loss in the enzymatic activity of the material.     

Crystallization and properties of L-arginine deiminase of Pseudomonas putida.

Crystalline L-arginine deiminase of Pseudomonas putida was prepared by the following steps: sonic disruption, ammonium sulfate fractionation, protamine sulfate treatment, DEAE-cellulose column chromatography, and L-arginine-Sepharose 6B chromatography followed by crystallization. This procedure yields a crystalline pure enzyme with a 45% recovery of the activity in crude cell-free extracts. The yield is significantly higher than that reported for this enzyme. The purified enzyme appears to be homogeneous in ultracentrifugation (s-o20, w equals 10.2 S) and isoelectric focusing (pI equals 6.13). The purified enzyme showed two bands on disc gel electrophoresis, both carrying out the deimination of L-arginine. Electrophoresis in the presence of beta-mercaptoethanol plus Na dodecyl-SO4 gave a single band (Mr, 54,000). Specific activity of this enzyme was 58.8 mumol of L-citrulline formed per min per mg of protein at 37 degrees. The optimum pH of the purified enzyme was 6.0 and maximal activity was obtained at 50 degrees. The molecular weight of the native protein was 130,000 by gel filtration and 120,000 by sedimentation-equilibrium measurements. The spectrum of the pure enzyme showed absorption maximum at 280 nm and the value of E-1%-1 CM AT 280 NM WAS 10.48 IN 0.05 M potassium phosphate buffer (pH 7.0). The crystalline enzyme hydrolyzed several L-arginine analogues. L-Homoarginine, L-alpha-amino-gamma-guanidinobutyric acid, and L-alpha-amino-beta-guanidinopropionic acid competitively inhibited the hydrolysis of L-arginine with Ki values of 25.7, 7.5, and 4.0 times 10- minus 3 M, respectively. p-Chloromercuribenzoate, Ag-+, and Hg-2+, and several metal ions inhibited the enzyme.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

The activating system of chitin synthetase from Saccharomyces cerevisiae. Purification and properties of the activating factor.

The yeast proteinase that causes activation of the chitin synthetase zymogen has been purified by a procedure that includes affinity chromatography on an agarose column to which the proteinaceous inhibitor of the enzyme had been covalently attached. The purified enzyme yielded a single band upon disc gel electrophoresis at pH 4.5 in the presence of urea. At the same pH, but without urea, a faint band was detected in coincidence with enzymatic activity, whereas at pH 9.5, either in the absence or in the presence of sodium dodecyl sulfate, no protein zone could be seen. From sedimentation and gel filtration data, a molecular weight of 44,000 was estimated. The proteinase was active within a wide range of pH values, with an optimum between pH 6.5 AND 7. Titraton of the activity with the protein inhibitor from yeast required 1 mol of inhibitor/mol of enzyme. A similar result was obtained with phenylmethylsulfonyl fluoride, an indication that 1 serine residue is required for enzymatic activity. The enzyme exhibited hydrolytic activity with several proteins and esterolytic activity with many synthetic substrates, including benzoylarginine ethyl ester and acetyltyrosine ethyl ester.A comparison of the properties of the enzyme with those of known yeast proteinases led to the conclusion that the chitin synthestase activating factor is identical with the enzyme previously designated as proteinase B (EC 3.4.22.9). This is the first time that a homogeneous preparation of proteinase B has been obtained and characterized.     

Pyruvate kinase from human skeletal muscle

A simple method is described for the isolation of crystalline pyruvate kinase from human skeletal muscle. The enzyme was purified by ammonium sulfate fractionation, heat treatment and crystallization. Two crystal forms of pyruvate kinase differing in solubility but not in specific activity were found. The homogenous enzyme preparations in triethanolamine buffer, pH 7.6 reveal at 25 degrees a specific activity of 245 U per mg protein, and of 340 U/mg in potassium phosphate buffer (50 mM). The enzyme is activated by inorganic phosphate and fructosediphosphate to the same extent, and inhibited non competetively by ammonium ion. The molecular weight as measured by gel filtration is 220,000 daltons and the enzyme molecule is composed of 4 subunits.     

Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2.

The effects of various anions on decreasing the solubility of acidic Hypoderma lineatum collagenase at pH 7.2 and 18 degrees C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO4(2-)/H2PO4- > SO4(2-) > citrate 3-/citrate2- >> Cl-. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH.     

ABSORPTION OF PEPSIN BY CRYSTALLINE PROTEINS

Crystalline proteins, such as edestin or melon globulin, remove pepsin from solution. The pepsin protein is taken up as such and the quantity of protein taken up by the foreign protein is just equivalent to the peptic activity found in the complex. The formation of the complex depends on the pH and is at a maximum at pH 4.0. An insoluble complex is formed and precipitates when pepsin and edestin solutions are mixed and the maximum precipitation is also at pH 4.0. The composition of the precipitate varies with the relative quantity of pepsin and edestin. It contains a maximum quantity of pepsin when the ratio of pepsin to edestin is about 2 to 1. This complex may consist of 75 per cent pepsin and have three-quarters of the activity of crystalline pepsin itself. The pepsin may be extracted from the complex by washing with cold N/4 sulfuric acid. If the complex is dissolved in acid solution at about pH 2.0 the foreign protein is rapidly digested and the pepsin protein is left and may be isolated. The pepsin protein may be identified by its tyrosine plus tryptophane content, basic nitrogen content, crystalline form and specific activity.     

ISOLATION,CRYSTALLIZATION, AND PROPERTIES OF SWINE PEPSINOGEN

1. A method is described for the preparation of pepsinogen from swine gastric mucosae which consists of extraction and fractional precipitation with ammonium sulfate solutions followed by two precipitations with a copper hydroxide reagent under particular conditions. Crystallization as very thin needles takes place at 10°C., pH 5.0 and from 0.4 saturated ammonium sulfate solution containing 3–5 mg. protein nitrogen per milliliter. 2. Solubility measurements, fractional recrystallization, and fractionation experiments based on separation after partial heat or alkali denaturation and after partial reversal of heat or alkali denaturation failed to reveal the presence of any protein impurity. 3. The properties of the enzymatically inactive pepsinogen were studied and compared with the properties of crystalline pepsin. The properties of pepsinogen which are similar to those of pepsin are: molecular weight, absorption spectrum, tyrosine-tryptophane content, and elementary analysis. The properties in which they differ are: enzymatic activity, crystalline form, amino nitrogen, titration curve, pH stability range, specific optical rotation, isoelectric point, and the reversibility of heat or alkali denaturation. 4. Conversion of pepsinogen into pepsin at pH 4.6 was found to be autocatalytic; i.e., the pepsin formed catalyzes the reaction. Conversion of pepsinogen into pepsin is accompanied by the splitting off of a portion of the molecule containing 15–20 per cent of the pepsinogen nitrogen.     

Crystallization of salt-free chymotrypsinogen and chymotrypsin from solution in dilute ethyl alcohol

Chymotrypsinogen and chymotrypsin crystallize readily from dilute solutions of ethyl alcohol in the absence of salts. The crystals formed in the presence of alcohol differ in appearance from those formed in the presence of ammonium sulfate. Chymotrypsinogen yields well formed polyhedrons instead of fine needles usually produced in ammonium sulfate solution. Chymotrypsin yields fine needles in the presence of alcohol and rhombohedrons in the presence of ammonium sulfate. The enzymatic properties of the crystals formed in the presence of alcohol are identical with those of the crystals isolated in the presence of ammonium sulfate.     

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.     

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.     

Biochemical Studies on Yeast

The present authors obtained direct proof of the occurrence of glucose dehydrogenase in yeast. Optimum pH of the glucose dehydrogenation system in yeast was about 7.0. After dialysis of the salting out preparation, the dialysate revealed only a trace of activity. The addition of DPN or TPN restored the activity. The majority of the yeast glucose dehydrogenase precipitated below about 0.70 ammonium sulfate saturation, and there was no marked activity in 0.30 ammonium sulfate saturation. The yeast glucose dehydrogenase was observed to be highly specific for β-d-glucose.     

CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR : II. GENERAL PROPERTIES

A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ.     

Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver

Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.     

外来入侵植物空心莲子草几丁质酶特性研究

外来入侵植物空心莲子草因其强抗逆性而迅速蔓延,成为有害杂草.拟研究其病程相关蛋白--几丁质酶酶学性质,为探讨该植物的抗逆机理和入侵机制提供理论依据.空心莲子草经乙烯利处理后,其叶片蛋白质提取液在4℃经40～60%饱和硫酸铵溶液沉淀,SephadexG-75洗脱后得几丁质酶液.结果表明:空心莲子草几丁质酶最适反应pH为5.7左右,在pH4.0～8.0均表现稳定,当pH小于4.0和大于8.0时酶活力丧失50%以上,在pH2.0和10.0时没有活力;在30℃-80℃范围内,酶均具较高活性,最适反应温度为70℃左右,温度高于80℃后,酶稳定性下降;当底物浓度小于1.25%时,酶活力随底物浓度的增加而增加,在底物浓度大于1.25%浓度时,活力增加的趋势减缓.结果提示:也许空心莲子草的强抗逆性是因为其几丁质酶具较高的温度耐受性和较大的pH作用范围.     

Purification,Crystallization and Some Properties of Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s_20,w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66,000 from Polyacrylamide gel electrophoresis and to be 58,000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.     

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.     

STUDIES ON THIAMINE TRIPHOSPHATE II. THIAMINE TRIPHOSPHATE AS PHOSPHATE DONOR

Yeast hexokinase, muscle fructokinase and glucokinase and liverhomogenate have no activity of transferring phosphate from TTPto hexose. A fraction precipitated from yeast autolysate at0.5–0.7 saturation of ammonium sulfate rendered TTP totransfer its terminal phosphoric residue to ADP to form ATP. (Received November 25, 1961; )