Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed.     

Crystallographic refinement and structure of DNase I at 2 A resolution

The structure of bovine pancreatic deoxyribonuclease I (DNase I) has been refined at 2 A resolution using the restrained parameter, reciprocal least-squares procedure of Hendrickson and Konnert. The conventional R-factor for 16,104 reflections with I greater than or equal to 3 sigma (I) from 6.0 to 2.0 A resolution is 0.157. Bond lengths and angles of the refined structure are close to ideal values with root-mean-square (r.m.s.) deviations of 0.023 A and 1.4 degrees, respectively. The r.m.s. deviation of short non-bonded contacts from the sum of van der Waals' radii is 0.18 A. The orientation of side-chains shows a clear trimodal distribution of chi 1-angles at -60 degrees, 180 degrees, 60 degrees (in the order of preference) corresponding to staggered conformations. The chemically determined sequence was corrected at four positions, the major correction being an insertion of the tripeptide Ile-Val-Arg between Arg27 and Arg28. Extended hydrophobic regions in between, and on either side of, the two central six-stranded beta-pleated sheets are mainly responsible for the low average isotropic temperature factor of 11.9 A2 for the 2033 protein atoms. Besides the flexible loop region between Gly97 and Gly102 (Glu99 and Ser100 are disordered) and the carbohydrate side-chain, which both extend into a large solvent channel, only the exposed loop Arg70 to Lys74 shows elevated thermal mobility. The longest of the eight helices in DNase I, together representing 26% of the structure, has a 22 degree kink and consists of two alpha-helical segments (residues 136 to 144 and 145 to 155) separated by a 3(10)-helical turn. DNase I fragments 1 to 120 and 121 to 257 can be superimposed by an approximate 2-fold axis (r.m.s. deviation 1.49 A for 61 equivalent C alpha positions), suggesting that the enzyme might be the result of gene duplication. The two Ca2+ bound to DNase I under crystallization conditions are important for its structural integrity by stabilizing the surface loop Asp198 to Thr204 and limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102. The N-linked carbohydrate side-chain attached to Asn18 is of the high-mannose type with a branching point at the mannose residue in position 3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-dependent activity of human DNase I and its hyperactive variants.

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.     

Molecular evolution of shark and other vertebrate DNases I.

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca(2+)-binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high alpha-helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low alpha-helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild-type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.     

The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening. Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.     

Structure determination of histidine decarboxylase from Lactobacillus 30a at 3.0 A resolution

The crystal structure of histidine decarboxylase from Lactobacillus 30a has been determined by X-ray diffraction methods to a resolution of 3.0 A. This protein is a pyruvoyl-dependent enzyme that is formed by an unusual self-activation process. The structure was determined from an electron density map calculated using multiple isomorphous replacement phases from two heavy-atom derivatives and included contributions from anomalous scattering measurements. The final mean figure of merit was 0.79, based on 28,805 independent reflections. The molecule has an (alpha beta)6 subunit composition and crystallizes in the space group 14122 with a = b = 221.7 A and c = 107.1 A. There is one (alpha beta)3 half molecule per asymmetric unit. The (alpha beta)6 particle is dumbbell-shaped, with each (alpha beta)3 unit being approximately spherical, with a diameter of about 65 A. There is a large central cavity approximately 30 A deep around the molecular 3-fold axis of the (alpha beta)3 unit. The 3-fold related active site pockets are located around the bottom of this cavity and are separated from each other by a distance of approximately 23 A. The inner portion of each (alpha beta) unit, which lies near the interface between the two (alpha beta)3 particles, consists mainly of random coil with several small helical and sheet regions. The outer region of each (alpha beta) unit has an unusual structure consisting of two overlapping, predominantly antiparallel beta-pleated sheets, lined on each side by an alpha-helix. The walls of the central cavity are formed by the 3-fold repeat of two strands from this beta-sandwich structure and one of the helices.     

Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate

The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Expression and characterization of a DNase I-Fc fusion enzyme

Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.     

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition,catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.     

Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase.     

Crystallographic studies of the chicken gizzard G-actin X DNase I complex at 5A resolution

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.     

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction.

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular nuclease produced by a marine bacterium. II. Purification and properties of extracellular nuclease from a marine Vibrio sp.

Extracellular nuclease produced by a marine Vibrio sp., strain No. 2, was purified by salting out with ammonium sulfate and by chromatography on a DEAE-cellulose column and twice on a Sephadex G-200 column. The nuclease was eluted as a single peak in which the deoxyribonuclease (DNase) activity and ribonuclease (RNase) activity appeared together. Polyacrylamide disc gel electrophoresis showed a single band of stained protein which had both DNase and RNase activity. The molecular weight of the enzyme was estimated to be 100 000 daltons. When using partially purified enzyme from the DEAE-cellulose column, the optimum pH for activity was 8.0, and the enzyme was activated strongly by 0.05 M Mg2+ ions and stabilized by 0.01 M Ca2+ ion. These concentrations of Mg2+ and Ca2+ ions are similar to those of the two cations in seawater. Indeed, the enzyme revealed high activity and strong stability when kept in seawater. The presence of particulate matter, such as cellulose powder, chitin powder. Hyflosupercel, Kaolin, and marine mud increased the stability of the enzyme. When the hydrostatic pressure was increased from 1 to 1000 atmospheres, the decrements of the enzyme activity were more pronounced at 30 and 40 degrees C than at 25 or 50 degrees C. The enzyme activity was restored after decompression to 1 atm at 30 degrees C.     

Structure and function of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.     

Refined atomic model of glutamine synthetase at 3.5 A resolution

An atomic model of 43,692 non-hydrogen atoms has been determined for the 12-subunit enzyme glutamine synthetase from Salmonella typhimurium, by methods of x-ray diffraction including restrained least-squares atomic refinement against 65,223 unique reflections. At 3.5 A resolution the crystallographic R-factor (on 2 sigma data) is 25.8%. As reported earlier for the unrefined structure, the 12 subunits are arranged in two layers of six; at the interface of pairs of subunits within each layer, cylindrical active sites are formed by six anti-parallel beta strands contributed by one subunit and two strands by the neighboring subunit. This interpretation of the electron density map has now been supported by comparison with glutamine synthetase from Escherichia coli by the Fourier difference method. Each active site cylinder holds two Mn2+ ions, with each ion having as ligands three protein side chains and two water molecules (one water shared by both metals), as well as a histidyl side chain just beyond liganding distance. The protein ligands to Mn2+ 469 are Glu-131, Glu-212, and Glu-220; those to Mn2+ 470 are Glu-129, His-269, and Glu-357. The two layers of subunits are held together largely by the apolar COOH terminus, a helical thong, which inserts into a hydrophobic pocket formed by two neighboring subunits on the opposite ring. Also between layers, there is a hydrogen-bonded beta sheet interaction, as there is between subunits within a ring, but hydrophobic interactions account for most of the intersubunit stability. The central loop, which extends into the central aqueous channel, is subject to attack by at least five enzymes and is discussed as an enzyme "passive site."     

The relationship between human serum and human pancreatic DNase I.

Deoxyribonuclease (DNase) activities have been partially purified from human serum and pancreas. Several of their physical and enzymatic characteristics were determined and compared in order to evaluate their relatedness. Human serum deoxyribonuclease has an isoelectric point in the range of 3.9 to 4.3 and a molecular weight of 33,000 to 38,000. Optimal enzymatic activity at pH 7.0 was dependent on both Mg2+ and Ca2+, whereas a pH optimum of from 5.5 to 5.8 was observed in the presence of Mg2+ and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The proportion of single strand or double strand breakage products at early stages of DNA digestion were variable functions of the composition of the buffers employed for the reactions. Single strand break age was predominant under all reaction conditions. Double strand breakage occurred with greatest frequency under neutral conditions in the presence of Mg2+ and Ca2+, was inhibited by the inclusion of 0.15 M NaCl, and did not occur at pH 5.8 in the presence of Mg2+, EGTA, and 0.15 M NaCl. Human pancreas deoxyribonuclease exhibited essentially the same physical properties and enzymatic characteristics as those of the human serum enzyme. Thus, human serum deoxyribonuclease may originate in this pancreas.     

Crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation

The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5A resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid-opening mechanism.