Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Alternate g−g−. conformation for dinucleoside phosphates in solution

An alternative g^?g^? conformation (conformer I') for dinucleosides in solution has been deduced, based on potential energy calculations and nuclear magnetic resonance spectroscopy. This conformation is characterized by larger glycosidic torsional angles (X=94–111°) than those of conformer 1 (X=8–35°), although the other torsional angles are similar. There are thus four stable confonners (I, I', II and III) for dinucleosides in equilibrium with the open forms. The structure of conformer I' supports that of the ‘vertical’ double helix constructed by Olson (W.K. Olson. Proc. Natl. Acad. Sci. U.S.A. 74, (1977) 1775). Our data may suggest the possibility of interconversion between the vertical double helix and the regular double helix of A-form DNA, RNA or A'-form RNA.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Shape of the ciliary doublet microtubule in solution

The individual doublet microtubule (DMT) from Tetrahymena cilia had the appearance of a circular arc in solution and its contour length was around 5 to 6 μm as observed under a dark-field microscope. One end of the circular arc was exactly in focus on the slide under the microscope, whereas the other end was slightly out of focus, suggesting that the circular arc may be regarded as a part of a coiled helix. On measuring the contour length and the end-to-end distance of the arc in solution, we found that the Ca-induced transformation of the ciliary DMT took place at a Ca concentration of about 10^?6m in the medium; that is, the radius of the arc was 3.4 ± 0.9 μm at 10^?3m-Ca and 1.5 ± 0.1 μm at less than 10^?6m-Ca. Change in the pH of the medium brought about no significant difference in the radius of the arc of the DMT. The individual DMT of a negatively stained sample appeared to have a configuration similar to that of the circular arc in solution, bending at right angles to the partition wall between the A and B-tubules. Based on the criterion that the A-tubule is wider than the B-tubule, we found that the A-tubule occurred on the outer side of the circular arc in 90 to 100% of the samples.     

Transformation ofSchizophyllum commune: An analysis of specific properties

Several properties of transformation in the basidiomycete,Schizophyllum commune, were examined. The transformation efficiency of protoplasts made from germinating basidiospores is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 μm in length) or from mycelium. Transformation frequencies of 1000 transformants/μg of control plasmid DNA and 10⁷ protoplasts are sufficient for obtaining transformants with 2 × 10⁷ protoplasts and 10 μg of bank DNA from a genomic plasmid library. The probability of cotransforming with two plasmids is dependent on the DNA concentrations of each; concentrations can be adjusted to yield nearly 100% cotrasformants. The presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments ofSchizophyllum genomic DNA are used as the nonselected DNA. Transformation of aSchizophyllum protoplast does not require its fusion to another protoplast.     

Mass and molecular weight of isolated nuclear rings

Nuclear rings are cell structures found at the nuclear cortex wedged between the nuclear envelope and the chromatin fiber network. In previous publications we have dealt with their morphology, relationships with the nuclear membranes, chromatin fibers and cytoskeletal filaments; and more recently, with their measurements at high electron microscope resolution. In this article we have calculated the mass and molecular weight of 336 isolated nuclear rings from human circulating lymphocytes using a photometric procedure and polystyrene latex spheres as the standard for weight calibration. Our results show a range of mass of 0.4–35.5 × 10⁻¹⁶g (equivalent to 0.2–21.2 × 10⁸ Da with a positively skewed distribution (median: 3.3 × 10⁻¹⁶g or 2.0 × 10⁸ Da). Mass and volume of nuclear rings were highly correlated. In addition, it was possible to calculate the area, the whole mass and the mass per unit area of the nuclear envelope present in the center of the nuclear rings. The mass of this area also shows a lognormal distribution (median of mass/unit area: 37.3 × 10⁻⁸ pg/nm² or 1.9 × 10⁵ Da/nm²). We discuss the significance of this results as parameters for the characterization of the nuclear rings and their possible implications for a new interpretation of nuclear cortex architecture, nucleocytoplasmic traffic and macromolecule segregation between the two main cell compartments.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Interaction between the adenovirus DNA-binding protein and double-stranded DNA.

DNA-binding protein was characterized by previous investigators as a single-stranded DNA-binding protein analogous to the gene 32 protein of phage T4 (Van der Vliet &; Levine, 1973; Sugawara et al., 1977). In the studies presented here the interactions between natural and synthetic polynucleotides and the DNA-binding protein of adenovirus 2-infected HeLa cells have been examined. Polynucleotide melting techniques revealed a tight yet dissociable binding to the helix structure of double-stranded DNA. In addition, binding and filter binding competition experiments at high DNA to protein ratios revealed a specific binding to double-stranded DNA termini with a dissociation constant of 1 × 10^?9 to 2 × 10^?9m. The ability of DNA-binding protein to bind to heat-denatured viral DNA was confirmed but the binding to double-stranded DNA termini was more specific on a molar basis. DNA-binding protein can recognize both flush and staggered ends of double-stranded DNA molecules.     

Electron microscopic visualization of RecA-DNA filaments: Evidence for a cyclic extension of duplex DNA

As visualized by electron microscopy, RecA protein binds in a highly cooperative manner to single-stranded fd DNA in solutions of 0.01 M Tris (pH 7.5). The resulting nucleoprotein filament loops are 1.25 μm in length, have a fiber diameter of 12 nm and show an indication of a 4.5 nm repeat along the axis of the compact fibers. RecA binds to linear duplex fd DNA in solutions of 0.01 M Tris (pH 7.5) to yield chains of beads which, in the presence of Mg²⁺ and ATP, coalesce into smooth filaments with a length of 1.9 μm (the length of protein-free fd duplex DNA) and have a fiber diameter of 12 nm. In solutions containing Mg²⁺ and ATP-γ-S, however, RecA binds to duplex DNA in a highly cooperative manner to yield rigid filaments 3.0 μm in length. These filaments are 12 nm in diameter and show a very clear 7.5 nm axial repeat. This extension of DNA to 150% of its usual length in the apparent absence of any single-stranded components suggests that the DNA helix must also be highly unwound and provides new insights into the mode of RecA action.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Model calculations on the kinetics of oligonucleotide double helix coil transitions. Evidence for a fast chain sliding reaction

Relaxation data obtained previously for the double helix coil transition of oligoriboadenylates and oligoribouridylates are compared to the results of numerical calculations according to various models. In these models the helix coil transition is described by individual rate constants for the first steps of helix formation, whereas the rate constants of the following steps of helix chain growth are assumed to be uniform. The existence of various helix intermediates containing the same number of base pairs is accounted for by statistical factors. First a quasistationary treatment of a zipper model is used for an analysis of the influence of various model parameters. Then relaxation spectra are calculated including helix coil intermediates explicitly without any assumption of quasistationarity. The relaxation spectrum calculated for any chain length N comprises N—1 fast processes with time constants in the range of 0.1 to 0.5 μs and one slow process with a time constant τ depending upon the nucleotide concentration (τ is usually in the ms time range). The fast processes are associated mainly with the unzippering at helix ends and are usually characterized by relatively small amplitudes, whereas the slow process represents the overall helix coil transition usually characterized by a very large amplitude.Consideration of staggered helix series (where the different helix scries are coupled to each other by the single stranded state) leads to a spectrum of slow relaxation processes with one separate relaxation process for each helix series. It is shown that this “non-sliding” staggering zipper model is not consistent with the experimental results. The measured relaxation curves can be represented by single exponentials for nucleotide chain lengths 8 to 11 (within experimental accuracy). This is also true for conditions where several, clearly separated time constants should be expected according to the theoretical model. The experimental data suggest the existence of a direct coupling between different series of staggered helices by a chain sliding mechanism with a time constant < 1ms. Chain sliding may be explained by diffusion of helix defects along the double helix such as diffusion of small loops. A simple model calculation for the diffusion of a bulge loop assuming quasistationarity suggests a sliding time constant around 100 μs for a helix comprising 10 base pairs.Finally some thermodynamic and kinetic parameters are evaluated according to the “sliding” staggering zipper model: The negative activation enthalpy observed for helix recombination can he described using a series of nucleation parameters indicating reduced stability constants for the first three base pairs. Nucleation may usually be achieved with the formation of the third or fourth base pair depending upon the magnitude of the chain growth parameter. The rate constant of helix chain growth is around 10⁶ s^?1 at 0.05 M [Na⁺] and increases to about 4 × 10⁶ s^?1 at 0.17 M [Na⁺].     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

A direct measurement of the unzippering rate of a nucleic acid double helix

The rate of double helix unzippering was determined directly by application of a fast temperature jump method to a nucleotide system of partly unzippered helices formed from oligoriboadenylates and oligoribouridylates of equal chain lengths (14 and 18 nucleotide residues). These helices showed a relaxation process in the time range of 0.1 to 0.3 μsec, that is assigned to the unzippering reaction. Measurements at 0.05 M and 0.1 M [Na⁺] demonstrated a rather small dependence upon the ionic strength. Increase of temperature increases the rate of unzippering. Simulation of the unzippering relaxation by a zipper model yielded a rate constant of base pair formation adjacent to a helix sequence of 8 × 10⁶ sec^?1 at 25°C associated with an activation enthalpy of 4 $\text{kcal}\text{mole}$. This elementary rate constant is higher than that obtained from a simulation of the overall recombination and dissociation rates of entire helices. The difference is attributed to reduced electrostatic and steric hindrance effects for base pair equilibration at helix ends.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Constancy of a 200 Å fiber in human chromatin and chromosomes

The diameter of 200 Å for modal chromatin fibers in unstimulated and stimulated peripheral human lymphocytes remains constant within a range of 5% throughout the cell cycle (including metaphase). Fiber dry mass varies slightly more, but its average of 6×10^?16 g/μ. remains constant also. — The use of colchicine induces metaphase fibers to shorten and results in a fiber mass per unit length increase of up to twofold. This change does not appear to be related to any change in total chromosomal mass. Fiber shortening, consequently and, with it, shortening of the chromosome are considered to be essentially conformational events.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Theory of melting of the triple helix poly (A + 2U) for a 1 : 2 mixture of Poly A to Poly U

The Zimm-Bragg theory is extended to treat the melting of the triple helix poly (A + 2U) for a solution with a 1 : 2 mole ratio of poly A to poly U. Only the case for long chains is considered. For a given set of parameters the theory predicts the fraction of segments in the triple helix, double helix, and random coil states as a function of temperature. Four nucleation parameters are introduced to describe the two order–disorder transitions (poly (A + 2U) ? poly A + 2 poly U and poly (A + U) ? poly A + poly U) and the single order–order transition (poly (A + 2U) ? poly (A + U) + poly U). A relation between the nucleation parameters is obtained which reduces the number of independent parameters to three. A method for determining these parameters from experiment is presented. From the previously published data of Blake, Massoulié and Fresco⁸ for [Na⁺] = 0.04, we find σ_T = 6.0 × 10^?4, σ_D = 1.0 × 10^?3, and σσ* = 1.5 × 10^?3. σ_T and σ_D are the nucleation parameters for nucleating a triple helix and double helix, respectively, from a random coil region. σσ* is the nucleation parameter for nucleating a triple helix from a double helix and a single strand. Melting curves are generated from the theory and compared with the experimental melting curves.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.