Conformational stabilization of enzymes in covalent catalysis.

The enzymes aspartate aminotransferase, rhodanese, and chymotrypsin form covalent substituted-enzyme intermediates during the course of their catalysis. The present analyses show that, in these covalent intermediates, the enzyme proteins are stabilized against pH-induced structural transitions to inert forms that occur in the free enzyme species and other forms not covalently substituted.     

Inhibition of cell aggregation by specific carbohydrates.

One approach to investigating the potential role of surface carbohydrates in mediating intercellular adhesion is to study cell reaggregation in the presence of defined concentrations of specific saccharides. Fifteen different exogenously added saccharides were tested for their effect on the reaggregation of 24 h sea urchin embryo cells (Strongylocentrotus purpuratus) dissociated by removal of divalent cations. Aliquots (0.2 ml) of cell suspension were rotated at 68 rpm, 17 °C, pH 8.0, with varying concentrations (0.5 × 1^?1?0.5 × 10^?5 M) of the sugars. Relative percents of cell aggregation were determined using an electronic particle counter assay. In all experiments cell viability using trypan blue was over 95.8%. Among the sugars tested, in 15 separate experiments, d-galactose and N-acetyl-d-galactosamine consistently inhibited aggregation to the greatest extent at early time points. d-Galactose, at all concentrations tested, at 10, 20, 30, 40, and 60 min rotation, showed mean decreases of aggregation over control values in the absence of sugar of 59.3, 53.6, 43.2, 35.0 and 36.4%, respectively. N-Acetyl-d-galactosamine also caused mean decreases in aggregation of 73.5, 54.5, 40.8, 42.2 and 45.6%, respectively. Each difference over the control is significant to the p value of less than 0.01. In three experiments, β-galactosidase substantially inhibited reaggregation of these cells. These results suggest that galactopyranosyl-like groups may be implicated in mediating adhesion of 24 h sea urchin embryo cells to each other.     

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.     

Efficient protoplast isolation from fungi using commercial enzymes

Several commercial polysaccharases have been compared for their ability to liberate protoplasts from fungi. These enzymes were found to contain side activities capable of hydrolysing fungal cell walls. Protoplasts have been commonly isolated from fungi using enzyme systems prepared by workers in their own laboratories. However, these procedures are time consuming and considerable variation may be found between different batches of enzyme. The present study shows that high yields of protoplasts can be prepared from a variety of fungi using relatively cheap commercial enzymes. The yields obtained were normally as good as or better than those previously produced.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25 dihydroxyvitamin D3 in chick kidney and pancreas.

Cytosol fractions prepared from rachitic chick kidney and pancreas were analyzed for binding of vitamin D₃ metabolites by sucrose density gradient centrifugation. Both cytosol fractions were found to contain a 3.6S macromolecule which specifically binds 1,25-dihydroxy[³H] vitamin D₃ and in addition a 5 to 6S macromolecule which binds 25-hydroxy[³H]vitamin D₃. Sucrose gradient analysis of a KCl extract prepared from kidney or pancreas chromatin resulted in a peak (3.6S) of bound 1,25-dihydroxyvitamin D₃ which could not be distinguished from the cytoplasmic binding component. The interaction of 1,25-dihydroxy[³H]vitamin D₃ with the cytoplasmic binding component of both tissues occurred at low concentrations of hormone with high affinity.     

The specific and endogenous mitotic inhibitor of lymphocytes (chalone)

Aqueous extracts of various lymphoid tissues, but not of non-lymphoid tissues, contain a species-non-specific but cell-specific inhibitor of the transformation and DNA synthesis of PHA-stimulated human lymphocytes which is apparently not cytotoxic and is reversible. This activity is found in similar molecular weight fractions from pure lymphocytes obtained in culture and hence appears to be endogenous to the lymphocyte itself. This specific and endogenous mitotic inhibitor does not appear to be a result of competitive lectin-binding, thymidine pool size dilution, phosphorylation, destruction of thymidine, or the direct immunosuppressive effects of thymidine upon the lymphocytes themselves. Rather, it appears to be a result of the effects of a protein contained in the crude ultrafiltrate from lymphoid tissues whose properties correspond to those originally described by Bullough & Laurence for a ‘chalone’. The chalone activity from thymus appears to be specific for T cells rather than B cells.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

Developmental regulation of cytokinin, spore germination inhibitor discadenine and related enzymes in Dictyostelium discoideum

The amounts of discadenine (spore germination inhibitor of Dictyostelium discoideum) and its precursor, N⁶-Δ²-isopentenyladenine (i⁶Ade) in cells of D. discoideum were measured at various stages of differentiation. The activities of the enzymes involved in the biosynthesis of these bases, i.e., discadenine synthetase and 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase (5′-AMP isopentenyltransferase) in the cells were also measured. During differentiation, discadenine appeared in the cells at the stage of culmination before i⁶Ade was detected. The activity of 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase in cell extracts increased after the beginning of culmination, much later than the increase in discadenine synthetase activity. The order of appearance of these bases and enzymes is apparently the reverse of that expected from the biosynthetic route. The implications of these findings are discussed.     

Enzymatic methods for the determination of L-serine concentration and L-[14C]serine specific radioactivity in blood plasma

Methods are desribed for the use of l-serine dehydratase purified from Clostridium acidiurici for the determination of l-serine concentration and l[¹⁴C]serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of l-serine in standard solutions and in a commercially available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma l-serine concentrations in excess of 30 μm. The reverse isotope dilution method was used for plasma l-[¹⁴C]serine specific radioactivity measurements. Carrier l-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The l-serine was then converted to pyruvate with l-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding l-[U-¹⁴C]serine to plasma and comparing the experimentally determined l-[¹⁴C]serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 ± 0.8% (5) of this calculated value.     

Some radiometric microassays for butyrylcholinesterase.

The use of commercially available 5′-UTP-agarose as an affinity chromatography resin for RNase has been described. It was shown that at pH 5.3, 0.025 m piperazine-HCl buffer was effective for the adsorption of active RNase A and exhibited little nonbiospecific binding as has been shown earlier for SepharoseaPhpUp [Stewart, G. R., and Stevenson, K. J. (1973) Biochem. J.135, 427–441]. Phosphate buffer at either pH 3.0 or 5.45 eluted essentially all of the RNase activity added to the column; however, pH 5.45 was slightly more efficient. Competitive elution experiments with 2′(3′)-UMP yielded a linear plot of $\text{1}\text{(V ? V}{}_{\mathrm{o}}$ vs [I]. From this plot K_I and K_IM were calculated to be 70 and 130 μm, respectively. It is suggested that since this material is different from that which is used most often for RNase A affinity chromatography, it may prove useful for RNase binding studies.     

Effect of leucogenenol,a thymothyroid hormone,on the enzymes and several other constituents of the serum

It has been found that rats treated with the thymothyroid hormone, leucogenenol, have significantly elevated levels of alkaline phosphatase and lactate dehydrogenase in their serum 8 hrs. later. These levels remain elevated for 24 hrs. and 12 hrs., respectively. The concentrations of creatine phosphokinase and aspartate aminotransferase as well as total protein, albumin, calcium, inorganic phosphorus, glucose, urea nitrogen, uric acid and creatinine in the serum are not affected by treatment with leucogenenol. These results are in agreement with the previously reported findings that treatment with leucogenenol increases the rate of development of blood cells of the bone marrow, increases the rate of recovery from immunosuppression and enhances the immune response.     

Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein.

When ¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of ¹²⁵I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound ¹²⁵I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of ¹²⁵I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Hyperproduction of dihydrofolate reductase in Diplococcus pneumoniae by mutation in the structural gene: The absence of an effect on other enzymes of folate coenzyme biosynthesis

A unique group of mutations (ame^r) in the dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase, EC 1.5.1.3.) structural gene of Diplococcus pneumoniae determine a marked overproduction of the corresponding enzyme protein. Since findings with these mutations relate to a key metabolic function and may be important to the regulation of folate coenzyme synthesis in general, the same group of multations were also examined for their effects on a number of related enzymic activities. Mutant and wild-type cell-free extracts, in addition to dihydrofolate reductase activity, exhibited both dihydropteroate and dihydrofolate synthetic activities under the conditions employed. Four folate coenzyme-related enzyme activities could also be demonstrated with the same preparations. These are mediated by the following enzymes, serine hydroxymethyl transferase (l-serine: tetrahydrofolate 10-hydroxymethyl tranferase, EC 2.1.2.1), 5, 10-methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.5), 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) and glutamate formiminotransferase (N-formimino-l-glutamate: tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5). The ame^r mutations examined in the current study determined 3–80-fold increases in dihydrofolate reductase in comparison to the wild type. However, none of the other folate-related enzyme activities were altered. The possible significance of these findings in light of previous results is discussed.     

ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.     

NMR studies of P-450 model systems: new structural probes for sulfur-containing hemoproteins.

A simple model for ferrous cytochrome P-450 has been investigated by proton and carbon-13 Fourier transform NMR. In the proton spectrum of the β-phenethyl mercaptan-protoheme-CO complex, the protons α and β to mercaptide sulfur are observed 2.62 and 0.62 ppm upfield of tetramethylsilane. The ¹³CO spectra show characteristic shifts at 204.7 and 197.0 δ for neutral and deprotonated mercaptan complexes, respectively.     

A non-linear model for the synaptic basis of neuronal memory.

We propose a mathematical model for the synaptic basis of neuronal memory. The model incorporates non-linear effects in analogy with population growth problems of human beings, animals, biological species, crystal growth, etc., and provides a mechanism whereby the excitatory and inhibitory inputs produce alterations in a neurone which result in a long-lasting increase in transmitter release at a synapse.