Urease catalysis and structure: X. Alternate bonding-site isozymes of jackbean urease

Three previously uncharacterized, nongenetic urease isozymes have been analyzed by sucrose density gradient sedimentation, gel electrophoresis, and chemical reactivity. The full complement of isozymes could be reliably generated by choosing appropriate levels of NaCl, pH, and ethylene glycol, and was stable for several days in dilute solution. The three forms of interest were found to be quaternary isomers of other isozymes, but differed from them qualitatively in their bonding sites, with disulfide bonds being substituted for noncovalent bonds. The separation of these isomer-pairs during sedimentation and electrophoresis cannot be readily explained by differences in size or charge, but must rather arise from a difference in shape. A simple two-dimensional model can provide the appropriate molecular architecture to satisfy these requirements: Only one of the two half-units in each α-urease molecule undergoes disulfide bonding during polymerization, and it does so with two adjacent molecules, thus producing asymmetric polymers from symmetric starting components.     

Ethanol-induced cytochrome P-450: Catalytic activity after partial purification

Cytochrome P-450 was partially purified from liver microsomes obtained from control, ethanol, phenobarbital, and 3-methylcholanthrene-treated rats. Benzphetamine demethylation, benzpyrene hydroxylation and aniline hydroxylation activities were assayed in a reconstituted system using fixed amounts of reductase and lipids, and increasing amounts of cytochrome P-450 from each source. Cytochrome P-450 from ethanol-fed rats showed substrate specificity differing from cytochrome P-450 obtained from control, phenobarbital and 3-methylcholanthrene-treated rats.     

Lactoperoxidase-catalyzed iodination of surface membrane lipids

Lactoperoxidase-catalyzed iodination of intact cells, is known to label predominantly, if not exclusively, the exposed tyrosine residues of cell surface proteins. The present study demonstrates that during this iodination process surface membrane lipids are also iodinated through an enzyme-dependent step. Phosphatidylcholine, cholesterol-phosphatidylcholine liposomes and confluent secondary cultures of chick embryo cells were iodinated by the lactoperoxidase-glucose oxidase-glucose [¹²⁵I] procedure. Liposomes were efficiently labeled. In the cells, 20–30% of the radioactivity was found in proteins and 20–30% in the lipids. Both neutral and polar lipids were found to bind [¹²⁵I] covalently. Controls in which lactoperoxidase was omitted showed < 6% of the radioactivity found in liposomes or cells labeled with the enzyme.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Synthesis of a cytosine nucleoside of 2-amino-2-deoxy-beta-D-xylofuranose.

1-(2-Amino-2-deoxy-beta-D-xylofuranosyl)cytosine (13) was synthesized by three routes: (a) coupling of 2-deoxy-3,5-di-O-p-nitrobenzoyl-2-(trifluoroacetamido)-D-xylofuranosyl chloride (5) with 2,4-dimethoxypyrimidine and subsequent treatment with methanolic ammonia, (b) coupling of 5 with 4-N-acetyl-2-O,4-N-bis(trimethylsilyl)cytosine followed by treatment with methanolic ammonia, and (c) thiation of 1-[3,5-di-O-acetyl-2-deoxy-2-(trifluoroacetamido)-beta-D-xylofuranosyl]uracil (6) by treatment with phosphorus pentasulfide in pyridine followed by amination of the resulting 4-thionucleoside 12 with metanolic ammonia. The best yield was obtained via route (a).     

Appearance of lymphocyte surface markers and functional responses in neonatal and young rabbit spleens

We examined spleen cells from newborn to 1-month-old rabbits for easily detectable surface immunoglobulin, complement receptors, and for in vitro proliferative responsiveness to anti-immunoglobulin antisera and several mitogens. From birth through the first month of life about 15% of the cells from rabbit spleens had easily detectable surface immunoglobulin while about 45% had C3 receptors. In adults as many as 77% of the spleen cells had easily detectable surface Ig but the proportion with C3 receptors remained about 45%. The proliferative response to anti-allotype antisera was present at birth, and was at adult levels by 1 month of age. The proliferative response to pokeweed mitogen was low when cells were obtained during the first week of life but was comparable in magnitude to the response of adult cells by 2 weeks of age. In vitro responsiveness to concanavalin A was present at low levels at birth and increased sharply during the first week. We did not observe significant stimulation of spleen cells from neonatal to 4-week-old rabbits by lipopolysaccharide from Salmonella typhosa. Our data suggest that lymphocyte surface markers and functional responses appear asynchronously in spleen cells of developing rabbits.     

The role of subcellular organelles in hormone secretion : The interactions of calcium, vitamin A, vinblastine, and cytochalasin B in PTH secretion

To determine the role of subcellular organelles in hormone secretion, we studied the interaction of low calcium concentration (low Ca), retinol (vitamin A, vit A), vinblastine (VB), and cytochalasin B (CB) in parathyroid hormone (PTH) secretion. Bovine parathyroid tissue pieces were incubated in media containing the above agents. Vit A stimulated PTH release to a mean of 170% of control. This effect of vit A was diminished when tissues were simultaneously stimulated with low Ca and, furthermore, absent when tissues were pre-incubated in low Ca.VB had no effect on low Ca-stimulated secretion, but did inhibit vit A-induced secretion in the presence of low Ca.CB stimulated PTH secretion to a mean of 150% of control during the second and third hours of incubation. CB had at least an additive effect with low Ca in stimulating PTH secretion, with a more prompt and greater response than seen in normal calcium. VB did not inhibit the acute effect of CB on secretion in normal calcium media, but did inhibit CB-induced secretion during the third hour of incubation.None of the agents stimulated the release of lysosomal cathepsin D, and vit A and CB did not stimulate the release of LDH.Our results suggest that; (1) vit A and low Ca stimulate PTH secretion through a common pathway involving the cell membrane; (2) CB stimulates PTH secretion through a separate effect on the cell membrane or submembrane microfilaments, which normally retards secretion of PTH; and (3) microtubular proteins may facilitate basal secretion of PTH, but are not involved in low Ca-stimulated secretion of PTH.     

A new method for determination of elastolytic activity using (14C) labeled elastin and its application to leukocytic elastase.

A new, highly sensitive and specific assay for elastolytic activity is described which employs insoluble elastin randomly labeled with [¹⁴C]. The substrate was prepared by labeling amino groups of the protein in vitro with [¹⁴C] methyl groups by reductive alkylation. The substrate was used to quantitate elastolytic activity from human leukocytes and to compare leukocytic elastase with pancreatic elastase. Purified human leukocytic elastase was approximately one-fourth as active as pancreatic elastase. Similar difference between leukocytic elastase and pancreatic elastase activities was found when the enzymes were tested against succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, but not when t-BOC-L-alanine-p-nitrophenyl ester was used.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.