Philip L. Whitney

Affinity chromatography of carbonic anhydrase

Abstract An insoluble support for affinity chromatography of carbonic anhydrase has been prepared by coupling Sulfamylon (p-aminomethylbenzene sulfonamide) to Sepharose 4B. Carbonic anhydrase binds to Sulfamylon-Sepharose very strongly and can be eluted under mild conditions by the addition of enzyme inhibitors. The gel was used to purify carbonic anhydrase from human erythrocytes and to separate isozymes B and C. It was also employed to separate native enzyme from modified carbonic anhydrases. The apoenzyme and the carboxymethyl enzyme of human carbonic anhydrase B were both isolated by this method.

Particulate carbonic anhydrase in homogenates of human kidney

About 2% of human kidney carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been found in particulate fractions. Its distribution in the particulate fractions obtained by differential centrifugation suggests that it may be concentrated in the brush border. The particulate enzyme is like red cell carbonic anhydrace C in its susceptibility to inhibition by anions. Particulate carbonic anhydrase is firmly bound to the membrane and is not released by incubation at pH 10.6 and 37 degrees C or by addition of Triton X-100 or deoxycholate. In 10% Triton X-100 at pH 11.3 and 37 degrees C, the particulate enzyme is inactivated with a half time of about 20 min, and this is at least an order of magnitude slower than the inactivation of soluble enzymes in the presence or absence of membranes. The soluble enzymes are inactivated within a few minutes at 25 degrees C in 3-4% sodium dodecyl sulfate, but the particulate enzyme is relatively stable under those conditions, and its half-time of inactivation at 14 degrees C with a detergent-protein ratio of 25 was about 24 h. Gel filtration with Ultragel AcA-44 in sodium dodecyl sulfate indicates that the membrane carbonic anhydrase has a molecular weight of less than 66 000, so its stability is not due to association with large membrane fragments or vesicles. These results suggest that the membrane enzyme may be a different isozyme than the soluble carbonic anhydrases. Although present in relatively small amounts, its localization on the membrane could give it functional significance.

Use of cyanide and diethyldithiocarbamate in the assay on superoxide dismutases

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.

Possible Mechanism for Late Gestational Development of the Antioxidant Enzymes in the Fetal Rat Lung

We hypothesized that a possible mechanism to explain the significant increases that occur in the pulmonary antioxidant enzyme (AOE) system late in gestation might be an endogenous increase in the normal reactive O2 substrates for these enzymes. We found that lung O2 free radical formation increased approximately 175% between fetal day 18 and birth (p < 0.01). We also found that late fetal rat lung mitochondrial and microsomal rates of AOE substrate (H2O2) generation increased markedly, and there was also significantly increased lung lipid peroxidation products with increasing gestational age. These definite elevations in reactive O2 species production in parallel with the time course of maturational elevations in the pulmonary AOE system, suggest that increasing enzyme substrate concentrations could be a primary controlling mechanism for increasing lung AOE gene expression in preparation for birth of the newborn.

Comparative responses of premature versus full-term newborn rats to prolonged hyperoxia.

ABSTRACT: Because fetal rat lungs have lower baseline levels of both surfactant and antioxidant enzymes than full-term newborn rats, we questioned whether prematurely delivered rats might be more susceptible to O2 toxicity than those born at term. In the present studies, prematurely delivered rats (gestational d 21 of 22) and full-term rat pups were simultaneously put in >95% O2 after birth. Surprisingly, we found that the preterm rats were not more susceptible to O2-induced lung damage and lethality than full-term newborns, but, in fact, the composite percentage of survival was even greater in the preterm pups from 7 to 9 d in hyperoxia and were similar thereafter up to 14 d in high O2. In addition, the preterm rats showed significantly decreased lung wet/dry weight ratios and consistently less severe pathologic evidence of pulmonary edema compared with term rats at 6 and 8 d of O2 exposure. The premature pups demonstrated the capability of inducing pulmonary antioxidant enzyme responses to hyperoxia by 3 d, and had significantly elevated copper-zinc superoxide dismutase, catalase, and glutathione peroxidase activities (and lung surfactant contents) at 6 d of O2 exposure compared with the term rats in O2. The rates of lung total O2 consumption and cyanide-resistant O2 consumption at d 6 in hyperoxia were not different for preterm versus term pups. Although the basis for the transiently improved survival and decreased evidence of pulmonary O2 toxicity in the preterm rats is presently unknown, these findings clearly indicate that premature animals of at least one species are equally able to induce protective lung antioxidant enzymes and surfactant responses to hyperoxia as full-term newborn animals.

Negative Regulation of Antioxidant Enzyme Gene Expression in the Developing Fetal Rat Lung by Prenatal Hormonal Treatments

ABSTRACT: Prenatal administration of thyrotropin-releasing hormone (TRH) or TRH plus dexamethasone (DEX) to pregnant rats accelerates lung surfactant system development in late gestation, but paradoxically depresses the normal late gestational elevation in fetal lung antioxidant enzyme (AOE) activities (Pediatr Res 30:522, 1991). In these present studies, we tested whether both prenatal hormonal treatments act to depress normal fetal lung AOE development by negative regulation of AOE gene expression. We used solution hybridization to quantitate the concentration of AOE mRNA. Results of the developmental studies revealed significantly decreased lung mRNA concentrations of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase in late gestation as a result of prenatal TRH treatment. The addition of DEX administration did not reverse the lowered expression of lung AOE genes due to TRH treatment, but instead resulted in significant additional decreases in pulmonary AOE mRNA levels at both 21 and 22 d of gestation. The tested AOE mRNA half-lives (stabilities) revealed no significant differences between controls (8.0–10.5 h) and TRH-treated (8.2–9.5 h) and TRH-plus-DEX treatment (7.8–10.7 h) groups. These findings suggest that prenatal treatment with TRH and with TRH plus DEX acts to depress the normal late fetal lung AOE activity elevations by (direct) negative regulation of AOE gene expression, and the decreased AOE expression is likely regulated at the level of gene transcription rather than posttranscriptionally.

Microdetermination of inorganic sulfate using thin-layer plates

Abstract Inorganic sulfate was precipitated on cellulose thin-layer plates with the radioactive reagent, 133 BaCl 2 . Excess reagent was removed by repeated washings with an acidic BaCl 2 solution. The residual activity was transferred to vials by cutting out the point of application and its immediate surroundings. Counting was performed in a scintillation well γ-counting system. The concentration-activity curve was linear.

81Br-nmr studies of carbonic anhydrase☆

Abstract 81Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a 81Br line broadening which is determined by the lifetime of Br− bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a 81Br nmr line broadening. Cl − Br − competitive binding studies, using 35Cl nmr, suggests that the failure to observe 81Br broadening is due to an increase in the lifetime of a zinc bound Br−. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a 81Br nmr line broadening. Estimates of the lifetime of zinc bound Br−, τM, are 4 × 10−7 sec. for CAM-HCAB at pH 8 and 1 × 10−7 sec. for BCA at pH 7. The lifetime for Br− bound to HCAB is estimated to be ≥10−6 sec.

H2O2 Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H2O2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H2O2-mediated changes in CFTR activity were examined. In Ussing chambers, H2O2-induced anion currents were sensitive to the CFTR inhibitors CFTRinh172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 μM H2O2 were transient. Cyclooxygenase inhibitors blocked the H2O2 response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H2O2 responses. EP1 and EP4 agonists mimicked H2O2 in both control and MRP4 knockdown cells. Thus, H2O2 activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane.

Effects of acetylation and guanidination on alkaline conformations of chymotrypsin.

Guanidination leads to stabilization of several globular proteins, including bovine chymotrypsinogen, as determined by hydrogen isotope exchange (P. Cupo, W. El-Deiry, P.L. Whitney, and W.M. Awad, Jr. (1980) J. Biol. Chem. 255, 10828-10833). The present study examined the binding of proflavin to guanidinated, acetylated, and native chymotrypsins in order to compare conformational flexibilities. The order of decreasing alkaline stabilities of the catalytically active conformations of the different delta-chymotrypsin forms was guanidinated, native, and acetylated proteins; delta-chymotrypsin showed greater stability than alpha-chymotrypsin. In each case removal of calcium reduced the amount of the catalytically active conformation. The alkaline pH dependence for the decrease of the catalytically active conformation of guanidinated alpha-chymotrypsin could not be attributed to the titration of a single group, indicating that the alpha-amino group of isoleucine-16 is not the sole feature regulating the conformational transition for this derivative. At neutral pH values delta-chymotrypsin exists completely in an active conformation and the percentage of alpha-chymotrypsin in this form is only slightly lower. These differences from earlier results are possibly due to differences in buffers, calcium ion concentrations, and ionic strength. The rate of inactivation of guanidinated delta-chymotrypsin with methyl acetimidate was much lower than the corresponding rate for the native enzyme. This suggests that guanidination increases enzyme stability which in turn leads to a reduced accessibility of the alpha-amino group of isoleucine-16.