Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.     

Succinate thiokinase in pigeon breast muscle mitochondria

Succinate thiokinase has been purified from pigeon breast muscle. It has been confirmed that the enzyme is entirely specific for ATP, and Km is very high (approximately 0.8 mM). Activity in mitochondrial sonicates is low enough for it to be doubtful whether the enzyme can support citric acid cycle flux in the tissue. The enzyme appears to have an Mr of 80 000-100 000, and to have two unequal subunits. As determined by SDS gel electrophoresis one subunit certainly has an Mr of 40 000.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties.

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.     

Limited proteolysis of pig liver CoA synthase: evidence for subunit identity

The bifunctional enzyme CoA synthase can be nicked by trypsin without loss of its activities. The original dimer of subunit Mr approx. 61 000 yields fragments of Mr 41 000 and 22 000 as seen on gel electrophoresis in the presence of SDS, but the nicked enzyme retains the native Mr of 118 000. Further proteolysis occurs rapidly in the absence of protecting substrates. The N-terminal of native CoA synthase is proline, and proteolysis exposes glycine as a second N-terminal. This evidence strongly suggests that the subunits are identical.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.     

Isolation and characterization of an inhibitory subunit of the Mg2+-Ca2+-ATPase of Escherichia coli

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked.2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase.3. The molecular weight of the ATPase inhibitor is about 10 000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition.4. The inhibitory action is independent of pH, ionic strength or the presence of Mg²⁺ or ATP.5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation.6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.     

Existence of stoichiometric amounts of an intrinsic ATPase inhibitor and two stabilizing factors with mitochondrial ATP synthase in yeast

Two proteinaceous factors, 15K and 9K proteins, which acted together to stabilize the inactivated yeast F1F0-ATPase-inhibitor complex [Hashimoto, T., et al. (1984) J. Biochem. 95, 131-136] were hardly distinguishable from the sigma and epsilon subunits, respectively, of yeast F1-ATPase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. However, they were clearly distinguishable from these subunits by analyses of the sequences at their amino terminals and by immunoblotting combined with SDS polyacrylamide gel electrophoresis. The two stabilizing factors and an ATPase inhibitor existed in mitochondria in equimolar ratios to F1-ATPase. These three protein factors were not present in purified F1-ATPase or in F1F0-ATPase preparations, but remained in the mitochondrial membranes after extraction of F1F0-ATPase with Triton X-100. These observations strongly suggest that the two stabilizing factors and the ATPase inhibitor form a regulatory substructure of mitochondrial ATP synthase, in addition to the F1 and F0 subunits.     

Purification of the constituent enzyme fractions of mycobacillin synthetase.

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.     

Protein analysis of cardiac sarcolemma: effects of membrane-perturbing agents on membrane proteins and calcium transport.

Protein composition of cardiac sarcolemmal membranes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were observed to contain about 20 polypeptide bands ranging from 18000 to 200 000 dalton mass. Out of these, six bands were prominent and together comprised 57% of the membrane protein. When sarcolemmal membranes, phosphorylated by [gamma-(32)P] ATP in the presence of Ca(2+) or Na+ with and without K+, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 2.4, the band III region (Mr 105 000) of gels was found to contain active sites of monomeric Ca-ATPase and (Na,K)ATPase. Bands I (Mr greater than 200 000), II (Mr 150 000), III (Mr 105 000), and VI (Mr 47 000) were accesible to trypsin; the extent of proteolysis was dependent on the time of exposure to, and the concentration of, trypsin (i.e, ratio of sarcolemmal protein/trypsin). Addition of molar sucrose protected sarcolemmal proteins from the tryptic proteolysis. Calcium transport was reduced by the action of trypsin; the degree of reduction was influenced by the time of exposure of membranes to trypsin as well as the concentration of trypsin. (Mg,Ca)ATPase activity, on the other hand, was elevated moderately at lower concentration and reduced at higher concentration of trypsin. Treatment with phospholipase C cium transport and (Mg,Ca)ATPase activity; electrophoretic patterns were unaffected by this treatment. Addition of lecithin to phospholipase C treated membranes produced a moderate increase in calcium transport. Exposure to Triton X-100 (1%) specifically solubilized three protein bands (Mr90 000, 67 000, and 57 000), whereas exposure to deoxycholate (1%) preferentially solubilized high-molecular-weight proteins, including band III (Mr 105 000); Lubrol-PX (1%) caused nonspecific solubilization of proteins, although the extent of solubilization with Lubrol-PX was considerably less than with either Triton or deoxycholate.     

Stabilization of rat liver mitochondrial F1-adenosine triphosphatase during chloroform-induced solubilization.

1. Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components of F1-ATPase are released. The low concentrations of ATP or ADP required (5 microM) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were found to be less effective. 2. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction of F1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a molecular weight identical with that of a beta-subunit of F1-ATPase. 3. Dissociation of the F1-ATPase molecule could also be prevented by aurovertin. 4. Crude F1-ATPase solubilized by chloroform treatment can be further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 mumol Pi/min per mg protein and the enzyme was composed of five protein subunits (alpha, beta, gamma, delta, epsilon) with molecular weights 58 000, 55 000, 28 000, 13 000 and 8000, respectively. 5. Chloroform-released F1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.     

Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris

The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Membrane reconstitution in chl-r mutants of Escherichia coli K 12. VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles.

Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5% polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg-2+ dependent, insensitive to inhibition by N, N'-dicyclohexylcarbodiimide and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg-2+ are able to incorporate the purified ATPase only in the presence of 2-6 mM Mg-2+. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increased in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.     

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.     

Molecular organization of glutamate-sensitive, chemoexcitatory membranes of nerve cells. Physico-chemical characteristics of the glutamate-binding synaptic membrane proteins of the rat cerebral cortex

Some physico-chemical properties of glutamate-binding proteins solubilized from rat cerebral cortex synaptic membranes and purified by affinity chromatography were studied. Purified proteins were shown to be homogenous during SDS polyacrylamide gel electrophoresis (Mr 14000). The Scatchard plots for L-[3H]glutamate binding to the purified membrane proteins revealed the presence of one type of binding sites with Kd 800-1000 nM and Bmax 180-200 pmol/mg of protein. Ultracentrifugation of the glutamate-binding membrane protein in sucrose linear gradient demonstrated that the position of the protein peak depends on protein concentration, i.e. after dilution of the sample the protein peak is shifted from 28 000-30 000 to 12 000-15 000. The values of sedimentation coefficients decrease correspondingly to 2.1S. Presumably, these processes are due to dissociation of receptor macromolecules. The glutamate receptor is a glycoprotein-lipid complex made up of several low molecular weight subunits.     

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.