CELLULAR AND BLOCHEMICAL ASPECTS OF CYSTIC FLBROSIS Thesis fcrjthe Segree of M. S. a‘éiCfiiGfis‘é STATE UNEVERSITY JOHN MULLED i‘éé KERSON 1976 in) LIBRA R Y ENDING BY H ‘ HUAB & SUNS’ . snax smomv mc. . LIBRARY BINDERS ABSTRACT CELLULAR AND BIOCHEMICAL ASPECTS OF CYSTIC FIBROSIS BY John Munro Nickerson Three lines of investigation were used to obtain baseline data for further studies of the disease cystic fibrosis. Carboxypeptidase B-like activity from Cohn Fraction IV-l was purified using TEAB cellulose and Sephadex G-200. An 86.5 fOId purification with 3.99% yield was obtained. Possible serum isozymes of carboxypeptidase B were found. Using a modified method for phosphate determination, ATPase activities from human erythrocytes and mouse brain microsomes were measured with and without human serum proteins present. The serum proteins activated the ATPase activities, and possibly acted hetero- tropically. Rabbit trachea epithelium was treated with cystic fibrosis or control serum and was examined after various incubation times by phase contrast microscopy. The differences in response of the tissue to the two sera were significant after 15 to 20 minutes incubation. CELLULAR AND BIOCHEMICAL ASPECTS OF CYSTIC FIBROSIS B)’ John Munro Nickerson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1976 Copyright by JOHN MUNRO NICKERSON 1976 DEDICATION To my parents, Mr. and Mrs. E. W. Nickerson, and my fiancée, Kathleen O'Neill. ii ACKNOWLEDGMENTS I wish to express my thanks to my committee, N. S. Henderson, C. W. Smith, C. C. Sweeley, and W. W. Wells. I would also like to thank W. F. Howatt, G. Polgar, and C. Wortley for obtaining serum samples; the National Cystic Fibrosis Foundation, the Michigan National Bank Trust Fund, and the Department of Anatomy for research funds; and K. M. O'Neill and D. Schmuck for technical assistance. I would especially like to thank Dr. Henderson for her guidance and suggestions during this research program. iii TABLE OF CONTENTS LIST OF TABLES . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . INTRODUCTION LITERATURE REVIEW . METHODS . Preparation of Erythrocyte Ghost Membranes Preparation of Brain, Skin, and Lung ATPases ATPase Assay . . Phosphate Determination . Protein Determination . Carboxypeptidase 8- Like Activity Assay Method . . . Fractionation of Cohn Fraction IV- 1 . Rabbit Trachea Ciliary Assay Preparation of Tissue . Assay . . . . RESULTS . . . . . . . . . . . . . . CBLA Assay Validation . . . . CBLA Purification From Cohn Fraction IV- 1 . Phosphate Determination . Results of ATPase Assays Serum Fractions . . . Rabbit Trachea Ciliary Response to Sera . Incorporation of (3 H)- -g1ucosamine Into Rabbit Trachea in the Presence of Sera (Control, C/F and FCS) DISCUSSION . CBLA: Introduction . CBLA Assay and Purification . iv Page vi vii 24 24 25 26 27 28 28 28 29 29 29 31 32 32 32 39 52 59 7O 79 84 84 85 Page Phosphate Determination . . . . . . . . . . . . . . . . . . . 87 ATPase Assay Validation . . . . . . . . . . . . 88 The Effect of Serum Proteins on ATPase Activities . . . . . . 89 Rabbit Trachea Ciliary Bioassay . . . . . . . . . . . . . . 90 Future Experiments Using the Ciliary Bioassay . . . . . . . . 92 Incorporation Experiments . . . . . . . . . . . . . . . . . . 92 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . 96 LIST OF TABLES Table Page 1. Clinical Symptoms of C/F . . . . . . . . . . . . . . . . . 4 2. CBLA From Cohn Fraction IV-l Enzyme Purification Summary Sheet . . . . . . . . . . . . . . . . . . . . . . 44 vi LIST OF FIGURES Figure l. CBLA Assay Validation. Linearity of Hippuric Acid formation with time . . . . . . . . 2. CBLA Assay Validation: Linearity of enzyme velocity (uMol hippuric acid formed per hour) with enzyme concentration . 3. Elution profile of CBLA and protein concen- tration from the S x 80 cm column of TEAE cellulose . 4. Elution profile of CBLA and protein concen- tration from the 3.5 x 170 cm Sephadex 6-200 (3011111111 0 o o o o o o o o o o o 5. Elution profile of CBLA and protein concen- tration from the 2.3 x 100 cm column of Sephadex G—200 . . . 6. Estimate of phosphate concentration by determination of reduced phosphomolybdate . 7. Absorbance loss with time . . 8. Rate of optical density loss vs. phosphate concentration . . . . . . . . . . . . . 9. Optical density of reduced phosphomolybdate with ethanolamine at wavelengths from 500 to 750 nm . . . . . . . . . . 10. Optical density of molybdate, ascorbic acid and monoethanolamine compared to distilled water . . . . ll. ATPase Assay Validation: Linearity of product (Pi) release with time . . . . . . . . 12. ATPase Assay Validation: Linearity of enzyme velocity with enzyme concentration . . vii Page 34 36 38 41 43 47 49 51 54 56 58 61 Figure Page 13. Lineweaver-Burk plot: total ATPase of RBC ghosts with varying concentration of 30-50% cut of pooled human serum . . . . . . . . . . . . . . . . . . . 63 14. Lineweaver-Burk plot: total ATPase from RBC ghost membranes with varying concentrations of the 30-50% cut . . . . . . . . . . . . . . . . . . . . 65 15. Lineweaver-Burk plot: total ATPase from RBC ghost membranes with varying concentrations of the 30-50% cut . . . . . . . . . . . . . . . . . . . . 67 16. Lineweaver-Burk plot: Na+/K+ ATPase from RBC ghost membranes . . . . . . . . . . . . . . . . . . . . . 69 17. Lineweaver-Burk plot: ouabain-insensitive ATPase from RBC ghost membranes with varying concentrations of 30-50% cut . . . . . . . . . . . . . . 72 18. Lineweaver-Burk plot: ouabain-insensitive ATPase from RBC ghost membranes with varying concen- trations of 30-50% cut . . . . . . . . . . . . . . . . . 74 19. Lineweaver-Burk plot: total ATPase from mouse brain microsomes with varying concentrations of 30-50% cut . . . . . . . . . . . . . . . . . . . . . . 76 20. Expression of secretory and dyskinetic responses of rabbit trachea epithelium to sera with time 0 O O O O O O O O O O O O O O O O O O O 0 O 0 O O O 78 21. Incorporation with time of radioactive glucos- amine into TCA precipitable material . . . . . . . . . . 81 22. Incorporation with time of radioactive glucos- amine into TCA precipitable material . . . . . . . . . . 83 viii INTRODUCTION The subject of this thesis is cystic fibrosis, a lethal genetic disease of high frequency, for which the basic defect is not known. The results presented within serve as requisite background for further investigations which will lead to an understanding of the pathology of cystic fibrosis and the corresponding normal physiologic situation. The topics of investigation presented here are: carboxypep- tidase B-like activity in Cohn Fraction IV-l, the effect of serum proteins on ATPases, and the effect of serum on rabbit trachea epithelium in 21232, The results reported here on the first two topics are concentrated on the investigation of the normal physiological condition, and the results on the third topic were directed toward the comparison of the effects of cystic fibrosis and control sera. The reasons for the investigation of carboxypeptidase B-like serum activity was that a deficiency in the activity was suggested to be the basic defect in cystic fibrosis, and the question of possible existence of isozymes was raised. The basis for the investigation of the effect of serum pro- teins on ATPases was that ATPase inhibitor materials of high molecular weight were reported in cystic fibrosis sera, saliva, and conditioned media; however, the mechanism of action of the corresponding control materials (which appeared to activate ATPases) was not analyzed. The rationale for investigating the effect of sera on rabbit trachea epithelium was that the mechanism of the response of the ciliated epithelium to sera has not been elicited. For each topic recommendations for further experimentation are given. Some of those experiments are already underway, while others will be started in the immediate future. LITERATURE REVIEW C/F (cystic fibrosis) is a simple autosomal recessive disease affecting 1 in 2,000 Caucasians, with lower frequencies in other races. The final result of homozygosity of the gene for C/F is death at an early age. Based on data obtained in 1972 Warwick et al. (1975) showed that the mean age for survival of 50% of C/F patients treated at C/F centers was 18.1 years. The most common cause of death in C/F is cor pulmonale, resulting from progressively increasing pul- monary hypertension, whose antecedent is chronic lung disease. The cause of disease in the lung is attributed to the buildup of thick, viscous mucus on which microorganisms can rapidly grow. This buildup of mucus is also found in the GI (gastrointestinal) tract. A particu- larly significant GI problem is that the pancreatic duct in many patients becomes clogged, and this blockage results in the degenera- tion of the pancreas with concommitant deficiency of pancreatic enzymes in duodenal fluid (di Sant'Agnese and Talamo, 1967). Clinical symptoms are listed in Table 1. Despite the many and protean manifestations of C/F, one abnormality of C/F is present in all cases. di Sant'Agnese et a1. (1953) showed that the sweat of patients with C/F was invariably high in the concentration of sodium and chloride. Mangos et a1. (1967) Table l.--Clinica1 Symptoms of C/F.* Failure to thrive Shortness of breath Barrel chest Labored breathing Noisy respiration Muscular weakness Cyanosis Digital clubbing Enlarged liver - Cirrhosis Meconium ileus Distended abdomen Poor weight gain Lack of subcutaneous fat Pancreatitis Diabetes Salt depletion Absence of vas deferens Cervical polyps Increased viscosity of sputum *No one symptom is unique to C/F. attributed these high concentrations to the lack of reabsorption of electrolytes in the straight portion of the sweat gland duct. The available treatment of C/F is purely palliative. A rationale for more effective treatment awaits the discovery of the basic defect in C/F. Researchers have been actively pursuing the basic defect in C/F for many years. Investigations comparing mucins from C/F and control subjects have been carried out. Dishe et a1. (1959) were not able to find qualitative changes in the sugar contents of the mucins of C/F subjects, except that the fucose content was slightly elevated. However, Gugler et a1. (1967) showed that the properties of C/F and control mucins were different. Using salivary mucins, the electro- phoretic patterns on polyacrylamide gels were different. However, when EDTA (ethylene-diamine tetraacetic acid) was added to C/F saliva the pattern changed to that of the normal saliva, and when calcium was added to normal saliva the same pattern as with C/F saliva was obtained. Other researchers have investigated differences in serum factors by biophysical methods. Wilson et a1. (1975) distinguished C/F homozygotes, normals, and C/F heterozygotes using isoelectric focusing on thin layer polyacrylamide gels in a pH 5 to 10 gradient. Volumes of individual sera were applied to the gels with a standard- ized amount of IgG (300 ug). A band focusing with a p1 = 8.46 was found in C/F homozygotes and C/F heterozygotes that was absent in control sera. Another band with an isoelectric point of 5.48, found in control sera, was absent in the C/F homozygotes and C/F hetero- zygotes. Researchers have used rabbit trachea as a model tissue for simulating the effect of C/F serum in_vivg_on human trachea. Spock et a1. (1967) compared the effect of C/F and normal sera and plasma on rabbit tracheal epithelium. C/F sera were found to cause a dyskinetic beat in the rabbit tracheal ciliary movement. Seventy-five out of 75 C/F patients' sera gave the dyskinetic effect, while sera from none of the 75 patient controls with a wide variety of other diseases gave the dyskinetic effect. The test procedure was as follows. Rabbit ciliated tracheal epithelium was dissected from cartilage rings and was cut into small fragments. The tissue was maintained in 20% (V/V) rabbit serum and 80% (V/V) Medium 199 for 4 to 6 days. The assay was performed at room temperature by placing a drop of test serum on a piece of actively beating ciliated epi- thelium and observing the tissue under phase contrast microscopy at 550x. In attempts to identify the serum factor having the dyskinetic effect, serum from a C/F patient was fractionated on Sephadex 6-200, and dyskinetic activity was found in a fraction of at least 200,000 daltons and in a fraction which eluted between 60,000-156,000 daltons. Other researchers have used oyster gill tissue to investigate toxic factors in serum of C/F subjects. Bowman et al. (1969) reported that the time required for the oyster gill cilia dyskinetic response caused by serum of C/F patients and heterozygotes was signifi- cantly less than that caused by serum from control subjects. In 47 of 47 C/F subjects, in 19 of 19 heterozygotes, and in 2 of 64 control subjects, the time required for ciliary dyskinesis was less than 35 minutes. The oyster gill cilia test was performed by examination of a 3 x 3 mm piece of gill tissue in a hanging drop preparation under phase contrast microscopy. A CD+ (ciliary dyskinetic positive) response was characterized by the accumulation of debris on the cilia and gill mounds and arhythmic beating of cilia. These investigators also noted a considerable variation in response between differing gill preparations, a loss of CD+ response after samples were repeat- edly frozen and thawed, and a lack of repeatability in the time required for CD+ response using hemolyzed serum samples. Bowman et al. (1970) used the oyster gill ciliary inhibition assay to show that a cationically migrating fraction of serum from C/F subjects on starch gels at pH 8.6 caused a CD+ response. No other fraction from the starch gel caused the dyskinetic response. They also demonstrated that on gel filtration using Sephadex G-200 a fraction of sera from 2 C/F patients and l heterozygote corresponding to roughly 125,000 to 200,000 daltons caused a CD+ response. Proteins found in this fraction included IgG (immunoglobulin G), IgA (immuno- globulin A), ceruloplasmin and haptoglobin. After DEAE (diethyl- aminoethyl) cellulose chromatography of serum from C/F, normal, and C/F heterozygotes, fractions containing mainly IgG as shown by immunoelectrophoresis were isolated and compared for oyster gill ciliary dyskinesis. The fractions from C/F and heterozygote serum caused CD+ response, while control sera fractions caused no dys- kinesis. Wood and di Sant'Agnese (1973) reported on the reliability of the bioassays for the C/F factor. They sent coded samples of C/F patient sera, heterozygote sera, and control sera to three research laboratories where the rabbit trachea or oyster gill cilia test was routinely used. The sera were prepared and shipped according to the individual laboratory's requests. Sera was stored at -30°C or on dry ice prior to and during shipment. No laboratory reported results that showed significant differences between C/F and control sera when Wood and di Sant'Agnese broke the code and analyzed the data. Doggett and Harrison (1973) reported that 43 to 500 units of heparin per ml of secretion can prevent saliva of C/F and heterozygote subjects from causing ciliary dyskinesis on oyster gills. The addi- tion of 1,000 units of heparin per ml of whole blood prevents ciliary dyskinesis, but after incubation overnight, the plasma regains its ciliary dyskinetic activity. Researchers have attempted to demonstrate that explants of rabbit tracheal epithelium to which C/F serum is applied serve as an accurate model of the properties of C/F subject tracheal epithelium 12.3322: Yeates et al. (1975) measured the rates of transport of albumin microspheres (0.5 u mean diameter) coated with radioactive (99Tc)-pertechnetate through the major air pathways. The progress of the radioactive spheres was monitored by a gamma camera or a scanning scintillation probe. The transport rates were tested for correlation against lung function tests, ciliary dyskinetic activity of the same patients' sera, Schwachman score, and clinical history. High ciliary dyskinetic activity and high maximum mid-expiratory flow rate correlated with high values of mucociliary tracheal transport rates. In the performance of the ciliary dyskinesis assay, they noted that material was discharged prior to the interruption of ciliary rhythm. They suggest that the discharge of material is related to the abnormal mucus secretion in C/F subjects in_vivg, Conover et al. (1973a) modified the rabbit ciliary technique of Spock et al. (1967) to produce a technique giving higher reli- ability in the detection of CD+ response. The modifications were: first, maintenance of a 37°C temperature while the assay was per- formed; second, prior screening of tissue for use in the assay; and third, fast set-up and handling of the tissue to minimize tissue trauma. Conover et al. reported 12 out of 12 ciliary dyskinesis positive sera from C/F patients and 2 out of 29 ciliary dyskinesis positive sera from the control group. Conover et al. also suggested that the ciliary dyskinesis factor is a normally present molecule which is over-produced in C/F, not properly inactivated, or inhibited in C/F. Conover et al. (1973b) cultured leukocytes from 3 C/F patients, 2 heterozygotes, and 4 controls. They found that the con- ditioned medium from leukocytes of the 3 C/F patients and the 2 heterozygotes caused ciliary dyskinesis, but that none of the controls' conditioned media caused ciliary dyskinesis with rabbit trachea. The conditioned media from long term lymphoid lines from 3 C/F patients and from 4 heterozygotes also produced ciliary dys- kinesis; but in 2 lymphoid lines from controls, there was also a 10 ciliary dyskinetic response from the conditioned medium. Rabbit anti-human IgG added to ciliary dyskinetic positive medium (incubated overnight at 4°C and centrifuged to remove the precipitate) caused the removal of the ciliary dyskinetic positive response from the super- natant solution. Beratis et a1. (1973) cultured skin fibroblasts from C/F patients, heterozygotes, and controls. When conditioned media were tested for ciliary dyskinesis by the rabbit tracheal ciliary test of Conover et al. (1973a), 1 out of 16 control subject media were CD+, 13 out of 13 C/F patient media were CD+, and 6 out of 6 heterozygote media were CD+. Beratis et al. also cultured amniotic fluid cells and found one CD+ medium in 10 control amniotic cell lines and in 4 out of 4 amniotic cell lines in which both parents' sera were CD+. They reported that the addition of IgG was necessary to obtain CD+ results in all fibroblast lines, but that the addition of IgG had no effect on the ciliary dyskinetic negative fibroblast lines. Rabbit anti-Human IgG treated media gave all ciliary dys- kinetic negative results. By fractionation of ciliary dyskinetic positive media using Amicon ultrafiltration, a fraction of approximate molecular weight of 1,000 to 10,000 could be obtained to which IgG was added caused a CD+ response. A similar fraction of ciliary dyskinetic negative media to which IgG was added did not give a ciliary dyskinetic positive response. Cheung and Jahn (1976a) reported that there is no change in the shape of a rabbit tracheal cilium throughout its forward and return bowing motion. They observed cilia via high speed 11 microcinematography and documented that the cilia penetrate into the mucus layer for 5-8° of the arch through which the cilium moves (a total of 35-40°). They also demonstrated that the cilium beat is more vigorous in the cephalic direction than in the return stroke. Using the same methods Cheung and Jahn (1976b) reported that by studying high speed microcinematography, C/F serum has no effect on the rhythm or the beat pattern of rabbit trachea ciliated epi- thelium. They reported that the C/F serum causes the expulsion and release of cells and debris from the epithelium and that so-called dyskinesia is a secondary effect of the C/F serum caused by drag forces applied to the functioning cilia. Satir (1975) found that the addition of calcium with ionophore A23187 arrested the lateral cilia of mussel gills. Neither calcium alone nor A23187 alone caused ciliary arrest. Satir suggested the hypothesis that a local rise in internal calcium concentration causes ciliary arrest. Bowman et a1. (1973) fractionated conditioned medium of fibroblast cultures from C/F patients and heterozygotes on DEAE cellulose and Sephadex 6-200. Three fractions were obtained that exhibited a ciliary dyskinetic positive response. No IgG could be detected in any of the 3 fractions. These 3 fractions corresponded to molecular weights of approximately 13,000, 20,000 and 150,000 daltons. The molecular weight of the ciliary inhibitor from C/F fibroblast conditioned medium was estimated by Barnett et al. (19733) using sucrose density gradient centrifugation, membrane filtration 12 and gel filtration. About 500 ml of medium was passed through Sephadex 6-15, and the protein fraction eluting at the void volume was applied to a DEAE cellulose column. The fractions eluted from that column were concentrated by membrane ultrafiltration on a filter which retained material of molecular weight greater than approxi- mately 1,000 daltons. The concentrated fractions were tested for ciliary dyskinetic activity. An aliquot of the fraction causing ciliary dyskinesis was put on a 5-20% (W/V) sucrose gradient and was centrifuged. After dialysis, using 3,500 dalton retaining dialysis tubing, the fractions of the sucrose gradient were assayed for ciliary dyskinesis. The fraction exhibiting a CD+ response correlated to a substance of molecular weight of about 9100 daltons. On a calibrated Sephadex G-SOF column, the ciliary dyskinetic activity eluted at a volume corresponding to a molecular weight of 4,500 to 10,000 daltons. By assaying the eluants and retentates of ultrafilters having molecular weight retention of greater than 100,000, 50,000, 10,000 and 1,000 daltons, the ciliary inhibitor was estimated to have a molecular weight between 10,000 and 1,000 daltons. Barnett et al. (1973b) reported the fractionation of con- ditioned medium from C/F patient-derived fibroblasts on DEAE cellu- lose. The fraction containing CD+ material was treated with pepsin or papain. Both treatments removed ciliary dyskinetic activity. Other aliquots of CD+ material were treated for 15 minutes at tempera- tures of 25°C, 50°C, 80°C, and 100°C. Only the material treated at 25°C for 15 minutes retained the ciliary dyskinetic positive response. 13 Schmoyer et al. (1972) fractionated C/F heterozygote serum on DEAE cellulose equilibrated with 0.005 M Tris at pH 7.5 and found that the material responsible for ciliary dyskinetic activity did not bind to DEAE cellulose. The fraction of serum not binding to the DEAE cellulose was applied to a CM (carboxymethyl) cellulose column, and material was eluted in a stepwise fashion. The fraction causing ciliary dyskinesis was eluted only in the first stepwise eluant which contained 0.04 M Tris-phosphate at pH 7.5. This fraction contained .less than 2% of the original amount of protein. The first fraction of the CM cellulose eluate was subjected to isoelectric focusing on pH gradients of 7 to 9 and 3 to 10. On the 3 to 10 gradient the fraction exhibiting ciliary dyskinetic activity was between pH 8 and 9. When fractionated on the 7 to 9 gradient, the fraction exhibiting ciliary dyskinetic activity was resolved into three fractions. These three fractions yielded one single band which migrated.the same distance on polyacrylamide gels. When the isoelectric-focused fraction giving ciliary dyskinesis was tested for antigenicity against rabbit monospecific anti-IgG, -IgM and -IgA, only anti-IgG cross-reacted. Schmoyer et a1. concluded that the ciliary dyskinesis factor belonged to the IgG class or was bound to IgG. Herzberg et a1. (1973) showed that the oyster gill ciliary inhibitor did not behave as an antibody. A preparation of C/F IgG (the IgG fraction of C/F patient serum) was shown to cause a CD+ response. On double immunodiffusion with sonicated oyster gill ciliary epithelium, anti-ciliary epithelium, saline control, and the 14 C/F IgG fraction, there were two precipitation lines shown--two between anti-ciliary epithelium and ciliary epithelium. There was no precipitin line between C/F IgG and the ciliary epithelium. There was no alteration of the precipitin lines by the C/F IgG. Barnett et al. (1973c) reported that the ciliary inhibitor has an isoelectric focusing point (pI) of 9.1 to 9.3 and was associ- ated with IgGl. The inhibitor was obtained from conditioned media and purified on DEAE cellulose by the method of Barnett et al. (1973a). Danes et a1. (1973) presented evidence that the cystic fibrosis factor (CFF) is bound to immunoglobulin. Using the oyster gill cilia assay and used medium from C/F and normal fibroblasts, they noted that the factor had the following properties: had a molecular weight less than 5,000 daltons, contained no uronic acid, was pH and heat labile, was negatively charged, and was bound to either IgGl or IgGZ. They showed that the interaction of the CFF with the immunoglobulins was not an antigen-antibody reaction. Their method was to incubate CD+ medium with immunoglobulin fragments con- taining antibody binding sites. Then the immunoglobulin fragments were precipitated. The CFF activity remained in the supernatant. When the constant regions of immunoglobulins were incubated with CD+ medium and the immunoglobulin fragments precipitated, the CD+ activity was removed from the supernatant. Conover et al. (1974) presented evidence that suggested that the factor causing ciliary dyskinesia is a complex of the complement factor C3a and IgG. Beratis et a1. (1973) had previously shown that IgG was required to elicit ciliary dyskinetic activity and that 15 there was a factor of molecular weight between 1,000 and 10,000 daltons present in fibroblast media of C/F patients that was also necessary for dyskinetic activity. Conover et al. (1974) showed that the second factor was also present in the 1,000 to 10,000 molecular weight fraction of serum. They also showed that when con- trol serum, previously shown not to be CD+ was incubated with EACA (e-aminocaproic acid), the serum caused a CD+ response. The effect of EACA alone on rabbit trachea cilia was not noted in this paper. (EACA is known to inhibit anaphylatoxin inhibitor.) They also noted that when CD+ sera, either from EACA-stimulated or from C/F patients, were incubated with carbosypeptidase B (di-isofluorophosphate treated, highly purified) that the responses on rabbit trachea cilia changed to ciliary dyskinetic negative. Conover et al. (1974) tested purified C3a with IgG on rabbit trachea and reported a ciliary dyskinetic positive response. When an anti-C3a was added to C3a plus IgG, a slightly ciliary dyskinetic response was noted, and when the 1,000 to 10,000 molecular weight fraction and IgG were treated with anti-C3a, the same response was noted. Conod et a1. (1975) reported that sera from C/F patients caused more degranulation of lysosomal enzymes from sensitized PMN's (polymorphonuclear leukocytes) than the sera of control subjects. They also reported that heterozygote sera caused the degranulation of an intermediate amount of lysosomal enzymes. They tested sera from 5 control subjects, 7 C/F patients, and 8 heterozygotes. PMN's from healthy donors were sensitized with Cytochalasin B, a fungal 16 metabolite which prevents phagocytosis and allows lysosomes to open at the cell membrane when a phagocytic-inducing material is detected at the cell surface. The amount of lysosomal release was estimated by the measurement of activity of B-glucuronidase and myeloperoxidase released into the media surrounding the PMN's. Serum levels of both enzymes were measured and subtracted from the approPriate sample of PMN's treated with serum. Conod et al. suggested that the molecules which caused the greater amount of degranulation of PMN's by C/F sera might also cause excessive degranulation of exocrine gland cells, resulting in blockage of exocrine gland ducts. They also suggested that the PMN degranulators are the same as the ciliary inhibitors. Conover et al. (1973c) presented data that implicated anaphylatoxin inactivator as the site of the primary gene defect in C/F. They showed that in 3 C/F, 3 heterozygote and 3 control sera samples, there were no significant differences in the levels of com- plement components Cl through C9, whole complement, or C3 inactivator (by hemolytic complement assays). In tests of the amounts of Clq, C4, C3 and C3-proactivator by radial immunodiffusion, C3 was found to be elevated. These investigators did not indicate the level at which the differences were significant. They stated that commercial rabbit anti-human C3 will also cross-react with C3a or C3a bound to IgG, and suggested that high levels of C3a, not C3, were detected by radial immunodiffusion. Lieberman (1975) studied the hypothesis put forward by Conover et a1. (1974) that CBLA should be deficient in C/F patients and give rise to high C3a levels in C/F serum. (C3a was hypothesized 17 to be the cystic fibrosis factor.) Lieberman tested the levels of serum CBLA (carboxypeptidase B-like activity) in C/F patients, control patients with chronic lung disease, and healthy control subjects. He found no significant difference between C/F patients and healthy controls. But he found that patients with other chronic lung diseases had significantly higher levels of CBLA than healthy controls. In addition, Lieberman (1974) found that C3 levels in C/F patients were not significantly different from controls. He tested 14 C/F patients and 15 controls by radial immunodiffusion. CBLA in serum was characterized by Erdfis et al. (1964, 1967), and its function as an inactivator of C3a was described by Bokisch and Mfiller-Eberhard (1970). Erst et al. (1964) described a carboxypeptidase that cleaves C-terminal basic amino acids from polypeptides and hydrolyzes hippuryl-L-lysine, hippuryl-L-arginine, Hippuryl-L-ornithine, and N-acetyl-phenylalanyl-L-arginine. They found this carboxypeptidase B-like activity in sera of many animals, in human urine and thrombo- cytes, but did not find this activity in erythrocytes. The enzyme was activated by CoCl2 and, to a lesser extent, by NiSO It was 4. inhibited by EACA, benzoyl-L-arginine, and chelating agents. The optimum pH for CBLA varied according to the choice of buffer. But with most buffers tested, the optimum was between pH 7.0 and 8.5. Hippuryl-L-phenylalanine as a substrate gave no measurable activity with human serum, indicating to Erdas et al. that there was no carboxypeptidase A-like activity in serum. 18 Erst et al. (1967) partially purified the CBLA of human plasma by: first, precipitation between 30% and 55% saturated ammonium sulfate; second, dialysis and fractionation on DEAE-Sephadex A-50 with stepwise elution; and, finally, a second fractionation on a DEAE- Sephadex A-SO column using smaller increments of increasing salt con- centrations in stepwise elution. The purification achieved was about 250-fold. Many experiments have used fibroblasts in cell culture derived from skin biopsies of C/F and control subjects. Differences between C/F fibroblast cells and control fibroblast cells have been demonstrated to complement the differences indicated above with the conditioned medium from the fibroblast cultures. Barnett et al. (l973d) showed that conditioned media from fibroblasts of C/F patients caused ciliary dyskinetic positive responses. They also showed that the concentration of ciliary inhibitor increased linearly with increasing cell number and that the inhibitor continues to be produced in stationary phase C/F fibroblasts. Barranco et al. (1976) reported that C/F fibroblasts incor- porated only one half the tritiated thymidine that normal fibroblasts did. Heterozygote fibroblasts incorporated an amount between the 2 above quantities. Barranco et al. matched fibroblasts according to culture age, growth fraction, and doubling time. They tested the possibility that TK (thymidine kinase) of C/F and heterozygote fibro- blasts were lower than normal fibroblasts, suggesting that altered TK activity could account for the lower amount of thymidine 19 incorporated into DNA. Barranco et al. found the activity of TK to be the same in all fibroblast lines--C/F, heterozygote and control. Another finding was that C/F fibroblasts could not be synchronized by the excess thymidine block technique, while control fibroblast lines were quickly synchronized. These 2 results indicated to Barranco et al. that thymidine transport was altered. They further suggested that this hypothesis was consistent with other data indicating that membrane transport is abnormal in C/F. In a number of studies the transport of electrolytes and the electrolyte levels in C/F and control subjects' fluids and cells have been investigated. Blomfield et al. (1976) collected parotid saliva from C/F patients and child controls and compared the levels of calcium, potassium, sodium, phosphate, salivation flow rate, and amylase. 0f the above materials, only calcium concentrations differed signifi- cantly, with Ca2+ concentrations from C/F patients being the higher. Salivation in these studies was stimulated by 5% (W/V) ascorbic acid solution placed on the tongue every 15 seconds. Previous studies using lozenges or other materials to stimulate salivary secretion had given inconclusive results. Duffy et al. (1973) reported that C/F serum, plasma and IgG fractions have no increased capacity for binding Ca2+. Ten control serum samples and 9 C/F serum samples were used. It was shown that plasma from 8 patients with IgG myeloma had an elevated capacity to bind Ca2+. 20 Araki et al. (1975) demonstrated that C/F, heterozygotes and control sera differentially affect the resistance and the short circuit current of rat jejunum. SCC (short circuit current) is defined as the potential difference between the luminal side of the rat jejunum and the serosal side divided by the membrane resistance. It was shown that C/F serum caused the SCC to drop by almost a factor of 2, while heterozygotes' sera caused a drop by about a factor of 1.5. When C/F and control sera were heat treated (56°C for 30 minutes), there were no significant differences between SCC's of the two sera. There were no differences between heat-treated control sera and control sera not heat-treated. These effects were only noted when glucose was included in the bathing solution, and the effects on the SCC were greater when glucose was added only to the luminal side. Araki et al. suggested that the effect on SCC was due to glucose dependent Na+ transport. However, they stated that the stimulation of Na+ transport by glucose did not alter SCC. Preliminary experi- ments suggested that the effect of C/F serum added to the serosal side was less effective than the luminal. Rennert (1976) presented data which showed that RNA from C/F patient and heterozygote fibroblasts or leukocytes is undermethylated. He isolated RNA by phenol extraction from cells that had been grown in the presence of tritiated uridine and (14C-methy1)-methionine. Studies on the transport of methionine indicated that there was no difference between C/F and control fibroblasts. Rennert suggested that there is a defect in the metabolism of methionine, not involving the RNA methylases. He indicated that undermethylation was due to 21 overproduction of polyamines. Additional studies showed that there were statistically significant increases in spermidine and an uniden- tified polyamine product in C/F patient serum as compared to control serum. Rennert (1976) in conjunction with Mangos, has shown that O-methyl glucose transport in the rat jejunum is inhibited by spermidine added to control serum, by C/F patients' serum, and heterozygote serum when compared to control serum. Another experiment showed that C/F serum treated with a crude preparation of polyamine oxidases gave the same effect on the transport velocity of O-methyl glucose as control serum. Cole and Dirks (1972) reported that there were no significant differences between the ouabain-sensitive component of ATPase in erythrocyte ghost membranes from C/F and control subjects. They found a significant decrease in the activity of ouabain-insensitive ATPase from C/F erythrocytes when compared to control erythrocytes. In a second series of experiments, Cole and Dirks incubated normal erythrocyte ghost membranes with serum from control and C/F subjects. After a 16 hour incubation at 37°C, the ouabin-sensitive ATPase activity of the ghosts was determined. A significant decrease in activity of the C/F serum-treated preparation compared to the control serum-treated ghosts was noted. Cole and Sella (1975) presented data that saliva from patients with C/F causes a decrease in ouabain-sensitive ATPase of erythrocytes when compared to the effects of saliva from control subjects. They incubated saliva adjusted to isotonicity with 1.0 M NaCl plus human erythrocytes for 18 hours at 37°C. Then they prepared ghost membranes 22 from the incubated erythrocytes and assayed the membranes for ATPase activities. Ghost preparations incubated with C/F patient saliva 4. had significantly lower specific activities of Na+, K , Mg2+ ATPase, ouabain-sensitive ATPase and Ca2+ activated ATPase. In a second experiment, samples of C/F and control saliva were filtered in a Millipore filtration cell with a membrane filter that retained molecules of molecular weight greater than 1,000 daltons. To the retained material, water was added and the procedure was repeated until the phosphate concentration in the retentate of the saliva was less than 0.2 mM. The retentate was added to prepared erythrocyte membrane ghosts, and the ATPase activities were assessed. No significant difference was found between total ATPase activities of C/F and control retentate treated membranes. However, the ouabain-sensitive ATPase activities were significantly different with the C/F treated samples lower by almost 16% (P < 0.05). In 9 of 10 samples the ouabain-sensitive component was less. Ca2+ ATPase was not assessed in the second set of experiments. Schmoyer and Baglia (1974) reported that a factor was present in the conditioned medium from stationary phase fibroblast cultures from C/F subjects that resulted in decreased activities of human erythrocyte ghost Na+/l(+ ATPase, Mg2+ ATPase, and Ca2+ ATPase when compared to used medium of a normal fibroblast line. With both C/F and control conditioned media, the activities were higher than the activities of the ATPase when 0.02 M TES at pH 7.5 was added to the erythrocyte ghost membranes in place of the conditioned media. However, the activities of the ghost membranes were higher when unused 23 medium was added to the ghost membranes in place of the used media. From these data it cannot be ascertained whether there is an acti- vator and inhibitor, a differential inactivation of the activator, or some other form of action on the ghost ATPases. However, it is clear that the C/F media effect on the ghost membranes was different from the control media. METHODS Preparation of Erythrocyte Ghost Membranes Membrane ghosts were prepared by the osmotic hemolysis method of Hanahan and Eklolm (1974). Outdated whole blood, usually A posi- tive or 0 positive, was obtained from the Lansing Regional American Red Cross. Plasma and residual buffy coat were separated from red blood cells by the following steps. About 50 ml whole blood were mixed with 200 ml of 0.15 M NaCl, and the solution was centrifuged at 4°C for 10 minutes at 2000 X g. The supernatant and buffy coat were removed by suction aspiration. The above steps were performed 3 times. The washed red blood cells were lysed by resuspension in 11.1 mM Tris at pH 7.5. Two ml of the packed red blood cells were transferred to 50 ml centrifuge tubes and 10 ml of the 11.1 mM Tris buffer were jetted into the centrifuge tube and the mixture was briefly shaken on a Vortex mixer at setting 4. Then an additional 20 ml of 11.1 mM Tris buffer were added. The tubes were centrifuged at 40,000 X g for 20 minutes. This centrifugation was sufficient to bring down the red cell membranes. The supernatant solution was removed by suction aspiration and the membranes were resuspended in 11.1 mM Tris buffer as above. The ghost membranes were washed by this method until they were pearly white, taking three to four centrifugations typically. 24 25 The ghost membranes were stored at 4°C, if they were to be used within 2 days; otherwise they were stored at -70°C. Preparation of Brain, Skin, and Lung ATPases The microsomal fractions of brain, skin and lung tissue from mice of the CD-l strain were isolated by the method of Akera and Brody (1968, 1969). Tissue was homogenized with a Teflon pestle glass homogenizer in 5 ml HS (homogenizing solution: 0.25 M sucrose, 5.0 mM histidine, 5.0 mM disodium ethylenediamine tetraacetic acid, and 0.01 mM DTT (dithiothreiotol) at pH 7.0) plus 0.15% (W/V) sodium deoxycholate. Fifteen ml of HS were added to the homogenate, and the mixture was centrifuged at 11,000 X g for 15 minutes at 4°C. The supernatant was decanted and saved. The pellet was resuspended in 10 ml HS plus 0.1% (W/V) sodium deoxycholate and homogenized for about half as long as the first homogenization. The mixture was centrifuged at 11,000 X g for 15 minutes at 4°C. The resulting super- natant was combined with the first supernatant, and this solution was centrifuged at 100,000 X gav for 30 minutes at 4°C in a Type 40 rotor. The supernatant was discarded and the pellet was resuspended in RS (resuspending solution: 0.25 M sucrose, 5.0 mM histidine, and 1 mM disodium ethylenediamine tetraacetic acid at pH 7.0) plus 0.01 mM DTT. Care was taken to thoroughly disperse pellets to prevent aggregated microsomes from being spun down with faster sedimenting material in the following centrifugations. This was done by homogen- izing the pelleted material briefly in a Teflon pestle glass homogen- izer. The next centrifugation was performed to separate the micro- somal fraction from the mitochondrial fraction. The solution was 26 centrifuged at 20,000 X gav for 20 minutes. The supernatant con- taining the microsomal fraction was centrifuged at 100,000 X gav for 30 minutes. The pelleted microsomes were resuspended in NaI solution (2.0 M NaI, 2.5 mM Na EDTA, 3.0 mM MgC12°6H O, and 5.0 mM histidine) 2 2 plus NazATP to 2.0 mM and stirred gently for 30 minutes at 4°C. The resuspended material was effectively solubilized at 2.0 M NaI, and the solution was adjusted to 0.8 M NaI with 1.0 mM Na EDTA. The 2 mixture was centrifuged at 100,000 X gav for 30 minutes and the resulting pellet, containing material insoluble at 0.8 M NaI was found to contain the ATPase containing microsomes. The pellet was resuspended in Tris-EDTA (10 mM Tris and 1.0 mM Na EDTA at pH 7.5) 2 to wash out the previous solution and centrifuged at 100,000 X gav for 30 minutes. The pellet was resuspended in RS, aliquoted, and stored at '7ooc 0 ATPase Assay ATPase activity was estimated by determining the amount of Pi (inorganic phosphate) present at the end of an incubation period. A blank containing all reagents but omitting the enzyme or containing inactivated enzyme was also incubated and any P1 in it was subtracted from the first tube to determine the amount of Pi released. All assay tubes were incubated in a 37°C water bath. All reactants were added to each tube in an ice bath, and the reaction was initiated with the addition of enzyme. The tube was shaken on a Vortex mixer (setting #4), and the tube put in a 37°C water bath. The total volume in each tube was 1.0 ml. Each tube typically contained 100 pl (550 ug protein) RBC ghost membranes, skin microsome (33 ug Protein), 27 or lung microsomes (39 ug protein), or 50 ul brain microsomes (33 pg protein), 200 pl of 5X concentrated ATP solutions, 0 to 200 pl activator or inhibitor solution and 11.1 mM Tris at pH 7.5 to 1.0 ml final volume. Each tube contained final concentrations of 100 mM NaCl, 20 mM KCl, and 20 mM Tris at pH 7.5. ATP final concentrations were 1.5, 1.0, 0.75, 0.60, 0.50, 0.40, or 0.33 mM disodium adenosine triphosphate trihydrate. The tubes were commonly incubated for 20 to 40 minutes and the reaction was terminated by the addition of 0.4 ml of ice-cold 10% (W/V) TCA (trichloracetic acid). The tubes were immediately put in an ice bath and then centrifuged at 1500 X gav for 30 minutes at 4°C. One ml of the supernatant was transferred to a clean test tube and the concentration of P1 was determined as below. Phosphate standards of 0.30, 0.25, 0.20, 0.15, 0.10, 0.05 and 0.00 mM Pi were run with each set of assays. Phosphate Determination The P1 concentration was estimated spectrophotometrically via modification of the method of Ames (1966) and the method of Drewes (1972). To 1 m1 of TCA-deproteinized solution, 0.4 m1 of acid molybdate (4.4% (W/V) ammonium molybdate in 1.0 N H2S04) was added, then 0.4 ml of 8% (W/V) ascorbic acid was added. Within 30 seconds of the addition of ascorbic acid, 1.0 m1 of monoethanolamine in a syringe was jetted into the tube while it was being shaken on a Vortex mixer (setting #4). The absorbance of the sample was read at 580 nm within 2 to 3 minutes following the addition of monoethanolamine. 28 Protein Determination Protein concentration was determined by the method of Lowery et a1. (1951). BSA (bovine serum albumin) standards of 0, 40, 80, 120, 160, 200 ug BSA/ml were tested with each set of unknowns. Carboxypeptidase B-Like Activity Assay Method CBLA (carboxypeptidase B-like activity) was assayed by the method of Lieberman (1975) using HLL (hippuryl-L-lysine) as substrate. Buffer N (500 mM potassium phosphate and 750 mM NaCl at pH 8.0) was made 12.5 mM HLL. Fifteen one-hundredths ml of enzyme preparation (either serum, Cohn IV-l, or an aliquot of fractionated serum or fractionated Cohn IV-l) was added to 0.1 ml of Buffer N plus HLL. The mixture was briefly shaken on a Vortex mixer and incubated at 37°C in a water bath or in a 37°C room for 60 minutes. To the incubation mixture 0.25 ml of l N HCl was added to stop the reaction. Hippuric acid was extracted from HLL by adding 1.5 ml of ethyl acetate and shaking the tube containing the reaction mixture and, ethyl acetate on a Vortex mixer (setting #4) for 15 seconds. The tube was centrifuged for 2 minutes at 1,000 X g to separate the two phases. One ml of the upper phase containing the ethyl acetate solution was transferred to a clean test tube using a semiautomatic pipettor. The ethyl acetate was evaporated at 120°C for 30 minutes. The dried contents were resuspended in 1 ml of water and the optical density at 228 nm was read using a Beckman 24 spectrophotometer. 29 Fractionation of Cohn Fraction IV-l Forty grams of Cohn Fraction IV-l paste were suspended in 2,000 ml potassium phosphate buffer (50 mM at pH 8.0), and the solution was filtered twice through approximately 2,000 ml of packed TEAE (triethylaminoethyl) cellulose in a 24 cm Buchner funnel. TEAE cellu- lose was precycled by suspending the powder to 7% (W/V) in 0.5 N HCl, filtering, and resuspending to 7% (W/V) in 0.5 N NaOH, and washing on a Buchner funnel with equilibration buffer (50 mM potassium phos- phate at pH 8.0). Material was eluted from the TEAE cellulose with 2,000 ml each of 0.1 M, 0.3 M and 1.0 M NaCl in equilibration buffer. The eluant from the 0.3 M NaCl wash and the latter half of the 0.1 M NaCl wash was applied to a 5 x 80 cm column of TEAE cellulose and eluted with a linear 0 to 0.3 M NaCl gradient of 2,000 ml volume. Pooled fractions were concentrated using XM 100A Amicon ultrafilters. Ultrafiltration was carried out at 4°C and the concentrate was applied to a 3.5 X 170 cm column of Sephadex G-200. The eluant was equili- bration buffer. The active fractions were pooled and concentrated by ultrafiltration as before. The concentrate was applied to a second Sephadex G-200 column with dimensions of 2.3 X 100 cm and was eluted with equilibration buffer. The active fractions were lyo- philized. Rabbit Trachea Ciliary Assay Preparation of Tissue The ciliated epithelium used in the rabbit trachea ciliary assay was obtained from a New Zealand white rabbit weighing more than 30 7 pounds. The rabbit was killed by cervical dislocation and the trachea was exposed as rapidly as possible. The trachea was cut first at the carina and then at the larynx, and put in sterile Hank's Balanced Salt Solution. The excised trachea was typically 4 to 5 cm in length. All further steps were performed in a laminar flow hood using sterile technique. All media were warmed to 37°C before use. Surrounding serosa, fat, and debris were dissected away from the trachea. The trachea was rinsed with sterile Medium 199 by trans- ferring the tissue into and out of two 15 X 100 mm petri dishes con- taining the solution. The trachea was cut into much shorter cylinders containing two cartilage rings by using a #22 surgical blade. These rings were cut at the cartilage defect (posterior wall) and 5 or 6 pieces were put into 10 X 35 mm culture dishes containing Medium 199 plus 10% (W/V) FCS (fetal calf serum). The pieces of tissue were temporarily put in a 37°C, 5% (V/V) C02 incubator. The ciliated epithelium was removed from the cartilage rings by: first, pinning the tissue to a sterilized wax block using two 25 gauge needles; second, nicking the epithelium at one end of the tissue; third, pulling up on the ciliated epithelium with very fine dissecting forceps, while gently nicking between the epithelium and the submucosa with a #10 surgical blade. After the epithelium was dissected off, it was cut into 15 to 20 pieces of roughly 1 X 1 mm size. These explants were transferred to 10 X 35 mm culture dishes containing 1.5 ml of Medium 199 with 10% (V/V) FCS. The medium was changed 4 or more times in 48 hours. Explants were kept at 37°C and 5% (V/V) C0 in a water 2 jacketed incubator. 31 Assay An explant was transferred to an ethanol-cleaned coverslip, and excess culture medium was removed with a pasteur pipet. Two drops of test serum were put on the explant, excess serum was removed, and the coverslip was mounted on a hanging drop slide. The hanging drop preparation was put on a microscope stage, maintained at approxi- mately 37°C by an air curtain incubator. The explant was observed with a conventional phase contrast microscope at 200 X. Micro- cinematography was performed using the above method with a 16 mm film camera. When films were not being made, the explant was put on an ethanol-cleaned microscope slide and two drops of serum were added. The explant was observed under phase contrast with an inverted microscope. If the air curtain incubator was not used, the samples were kept in a 5% (V/V) C02 incubator and observed for 10-15 seconds at room temperature at 5 minute intervals for up to 30 minutes. Characteristics noted were: the appearance of secreted or expulsed debris at the ciliary border, the change from rhythmic to dyskinetic beating of cilia, and the stoppage of beating of cilia. RESULTS CBLA Assay Validation The method used by Lieberman (1975) to assay for CBLA was tested to check assay validity. In Figure 1, product formation (hippuric acid) was shown to be linear with time, up to 2 hours incubation at 37°C between 0.025 mg/ml and 0.1 mg/ml of enzyme preparation. In Figure 2, it was shown that the rate of reaction is linearly proportional to the enzyme concentration. CBLA Purification From Cohn Fraction IV-l The procedure used to purify CBLA from Cohn Fraction IV-l was given in the Methods section. Approximately 240,000 units (1 unit 1 equals 1 uMOl hippuric acid released per hour) of CBLA was applied to a 24 X 5 cm columm of TEAE cellulose in a Buchner funnel as a batch separation. The last 1,000 m1 of the 0.1 M NaCl eluant and all 2,000 ml of the 0.3 M NaCl eluant were pooled. This fraction contained 85.2% of the original activity and gave a fold purification of 1.95. This material was applied to a 5 X 80 cm column of TEAE cellulose. The elution profile is shown in Figure 3. One peak of CBLA is seen between fractions 97 and 121. A second peak of CBLA is seen between fractions 133 and 148. A peak of high protein concentration is seen centered at fraction 109. The protein concentration tailed off from a 32 33 .:owumuu:oocoo :25 8&2» EB: mm A: 2:. 35m: om .3 "dim: mu .nlv 35B: 2: .mov .5393 ma non xuw>fiuom mafia: 56H mo :ofluwummoum a saw: woven usocfl mm: ocflmxHuqauxpzmmw: .oafiu spa: :oflumapom efio< Unnamed: mo spuemoeug ”coupmuufim> samm< «Emu--.fi .mua 34 (lw/CIIOV OlUndle IOW") 1.0 1.5 2.0 TIME (HOURS) 0.5 35 .:0Humuu=oo -coo oexuzo umcwmmw wouuofim ego: H enamfim aw mocfifi any we ”mowuwooHo>v momofim och .cOHHmnucoocoo maxnco no“: muse: mom cashew vflow ownsmmw: Hoznv xufiuoHo> maxuco mo Aufinmocfla ”ceaumwflam> xwmm< ~2w mhcd mad mNod _ — _ (IW/Slan) ALIOO'IHA 37 $33.33 053.8 A: “cowuwnucoocoo 530.3 .AIU .ucmsfio as N.mH vQCMwucoo coauomhm :omm .pcofivmhm Humz : m.o ow o.o ummcua a no“: emaoso>oe use :Ezfioo ecu ou vofifimmm mm: :ESHoo Hoccsm poccosm may scum Hmwhoume wonxfimflv mo HE ooom mo :uumn < .omoHsfiaoo m._._>_._.o< as 109 121 133 145 157 169 181 193 97 FRACTION NUMBER 39 maximum of 4.6 mg/ml at fraction 109 to its lowest concentration of 100 ug/ml from fraction 177 to the end of elution. The fractions containing the second peak of CBLA activity (fractions 132 to 153) were pooled, concentrated by membrane ultra- filtration to 19 ml, and applied to a 3.5 X 170 cm Sephadex G-200 column. The elution profile of activity and protein concentration is shown in Figure 4. One major peak of CBLA is seen between frac- tions 88 and 106. One major peak of protein has a maximum of 1,200 ug/ml at fraction 72 and tails off to roughly 300 ug/ml at the end of elution (fraction 149). Fractions 92 through 101 were pooled, concentrated from 47 ml to 6 ml by membrane ultrafiltration and applied to a 2.3 X 100 cm column of Sephadex G-200. The material was eluted with equilibration buffer. The elution profile of that column is given in Figure 5. Protein eluted in one main peak between fractions 37 and 63. CBLA eluted in one peak between fractions 41 and 51. Fractions 43 to 50 were pooled and lyophilized. Table 2 summarizes the results of the CBLA purification from Cohn Fraction VI-l. The activities of aliquots of the pooled fractions shown on Table 2 were measured in the presence of 10'4 M CoClz, which acti- vated the enzyme. However, the activities of individual fractions shown on the elution profiles were not measured with CoCl2 to help minimize enzyme self-destruction. Phosphate Determination The procedure described in the Methods section was used for determining phosphate concentrations. Phosphate was allowed to 40 #3336 953:0 . filv ”cofiumpucoocoo :flououa .fiov .wouooafioo one: HE m.v mo mcowuompm .Hmmmsn cowuwunwfifisco no“: vousfio cam vofifimmm one: HE ma mcflamuow cesaoo Eu ow x m exp scum .P_>_._.U< FRACTION NUMBER 42 .000>0000 000000 .0000300 000 00000 000000 “0000000000000 0000000 .0000000 000 0000: 0000m .000000000 0003 HE 0.N mo 000000000 .0000:: 0000000000500 :00: 000000 000 000000 0:0 00 0000000 0003 00 o M0000000 000000 00 000 x m.m 0:0 0000 000000000 0>0000 000000000000 .000000 .oom1u x000:00m mo 000000 00 co: 2 m.m 0:0 000w 0000000000000 0000000 000 .:>_._.o< 55 63 FRACTION NUMBER 39 47 31 44 000000 0.00 000.0 00.0 000.0 000.0 00 mm 000-0 00 000 x 0.0 000000000 000000 0mg 000000 oom1u 0.00 000 0.00 000.0 000.00 00 00 so 000 x mm 0000 000000000 000000 000 000000 m000< as 00000 x00>0000 0000: 00000 00000> 000m 0000 0000000m 0000000 0000000 x00>000< .000:m x00000m 000000000000 00000m 01>0 00000000 0:00 000m a map mucomonmou pcwom comm .cowuw>uomno mo coaumfi>ov unmvcmpm oumofiwcw mama Hounm .coflpmnucmuzoo oumnmmocm .m> ea owm um xuflmcow Hmowumo .oumvnxaoeozmmonm vooavon mo cow» um:HEnopoc x9 newumnucooaou oumnmmocm mo mumsflummuu.o .mflm OPTICAL DENSITY AT 580 nm 0.7 0.6 0.5 0.4 0.3 0.2 0.1 47 MOLAR ABSORBANCE OF 2150 AIM/cm $1 012 6.3 PHOSPHATE CONCENTRATION (mM) 48 .HmuoEouonmoapoomm um cmexuom m :H a: own an mouscfie we mom venouwcoe mm: uoHoo mo mmoH one .:ofiuumm muonumz as» :« onNUflwcfi mm ocw5m~ocacpo can .vwom ownuoomm .oumvnxfios kum mo :ofiuflvvm map x9 :owumcwe -uouow oumgmmonm Mom woummoum mum: .m 25 m.o .~.o .H.o .o.o mo moamemm .oswu an“: mmoH ooamnuomn mmoH xufimcov Hmofiumo mo ovum--.w .mfim RATE OF DECREASE (—OD 580/MINUTE) .0014 F 51 I I 1 0 0.1 0.2 0.3 PHOSPHATE CONCENTRATION (mM) 52 In order to determine the wavelength at which reduced phos- phomolybdate has its absorbance maximum, a wavelength scan from 350 to 750 nm was performed. Samples of phosphomolybdate initially con- taining 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 mM Pi were prepared according to the Methods section. The results of the wavelength scans are given in Figure 9. The optical densities all increase as the wavelength increases from 500 nm to 750 nm. The results below 500 nm will be considered later. Since a complete absorbance peak was not found in the wavelength range scanned, the wavelength of the absorbance maximum could not be determined, but it was greater than 750 nm. The 0 mM Pi solution, treated for phosphate determination, was used as a sample for a wavelength scan against a reference sample of water. The scan is shown in Figure 10. There was very little absorbance from 550 to 750 nm; however, from 350 to 550 nm, part of a large absorbance peak was observed. Results of ATPase Assays The procedure used for the assay for ATPase activity was given in the Methods section. The assay procedure was checked for validity. The amount of Pi released up to 90 minutes after the initiation of the reaction was tested. The results are given in Figure 11. Four different concentrations of RBC (red blood cell) ghost membranes were tested, and sample reactions were terminated at 10, 20, 30, SO, 70 and 90 minutes. The reactions released Pi linearly with time through 90 minutes. The slopes of the lines were determined by least-squares fits. When the rate of release of P1 was 53 .mcmom on» you cow: was nouoeouogmouuoomm Emon-ofin=ow em cmsxoom < .oocouomon a mm onEmm an 25 0.0 may magma a: omn on com scum voccmom one: .coflpoom muonuoz ago a“ ma peeve one: mcfiemfimcmcuo use .wfiom ownuooma .opmwnxfioe wfiom seas: on .m 25 om.o can .m~.o .o~.o .m~.o .o~.o .mo.o .o.o mo mofimsww .E: om“ on com aonm mcumcofio>m3 um ocfiemHocmnuo cum: opmcprosozmmosm voosvon mo spamCov HmoquOuu.m .mam OPTICAL DENSITY 0 500 S4 550 600 650 760 WAVELENGTH (nmI 750 SS .onom Hfism < o.m an“: vocamom max o>nso umofl on» ofifixz onom Adam < o.H you voccmom mm: o>nso ecu mo cofipuom pnmfin one .Ec own on omn scum mm: :mom :uw:o~o>w3 0:9 .nouwz coHHHumfiv mo oucouomon m umcfimmm voccmum mm: .cofluoom muonuoz on» cw woumofivcw we :owumaflenomow oumzmmozm Rom coummonm onEmm zoflpmuucoocoo .m 25 o.o one .uoumz voHHHumwv op wonmeoo ocflemaocmcuoocoa can cfioa oflnuoomm .oumvnxflos mo xuflmcov Hmoflumo-u.oH .wfim OPTICAL DENSITY 2.0 r 1.8% I 1.6 1.4- 1.2- 1.0- 0.8 - 0.6 ‘- 0.4 ' V 0.2 O . 350 40 430 56 560 560 600 WAVELENGTH (nm) 650 760 750 S7 .xuw>wuom mo Houmo -flvcfi can we wouuon cam wocwauouov mm: oumsmmocm .\zv wofi mo somufleem gum: wopmcfleeou ecu oexuco mo sewuwcvm spa: voumflpflcfi mm: :ofluomon och .oanao> Hmcfim HE o.~ cw mocmnnEoE amonm 0mm HE H.o use .N.o .m.o .e.o :fimucoo umozoa on omofim umozwfi: Eonm mo>uso one .oswu Sufi: omwoaou fifimv posvonm mo xufiumocfiq ”cowumvwam> Ammm< ommmh<-u.HH .mflm 58 0.5 I. 0.4 - _ _ 3 2 o O E: 9mm .5. >.:me0 ._ Hmcwm HE o.H :H osxnco Hey :ofiumuucoucoo oexnco pmcfimmw wouuon one: Anso:\vom H02: op nouuo>coo one mufim moumsvm -ummoH xn vocwmpnov m unawam Eoum mo>uso ecu mo momofim och .:0wuwupcoocoo osxnco no“: xuwoofio> oexnco mo xufiumocflq ":oflumvflaa> xmmm< ommmh -n.m~ .mfiw 61 u SI. . uliuklhhr 322 20:23:02. .8 c.- 2. wE>NZw .Ev ZO_._.N2w .vd Md Nd Pd O 1 . 1 . o L mod L Ed [mpd IHflOH/OBSVBWEH Id 10W”) ALIOO'IEA 62 .ah< . +Nwz n oumuumpsm .cofiuumon pumum on xfie :ofiumnsocw any on wovum who: He :oflumummoum umonm 0mm mo Acfiouoam m1 ommv H: ooH .cofluoom mvocuo: on wcfivpooom woummoum mumocm wee can xmmm< .HE o.H mm: oasao> Hmcfim .coflumuucoocoo Hmwuficfi He\ws mn.m mm: coaumuu Icoocoo :fiopopm .uso wow-om H: com .all ..II . spa: mumocm 0mm mo ommmh< Have» "uon xusmuuo>mozocwa-u.mfl .mam 63 .25: m N F _m._. so“: mocmHnEoe umocm 0mm Eonm ommmh< Hmpou ”uon xuzmuuo>mozocwg--.efi .wflm 65 1P 1.6- l—IHHOH/Vocl 1owfi) ALIOO‘IEINL I1/mMI 1/[SUBSTRATEI 66 .mH unseen ea czonm one mecwfl mouescmupmeoq .eceon ma ohswfim :a me one mfiefiueuee ece meocuos Hecpo .eom: we: 2 cofleueeoa 3.9% one .3 com AI .. I .3 :3 8N .fiI ..| .3 3: 03 All .3 :3 OS .Tullé :3 om; ..... .3 “use eom-om o: .enlnuu.ov "mfloeeam .uso eom-om can we meoMueHuceocoo wcwxue> no“: moceunaos amonm 0mm Eoum omemh< Heuou "uon xusmuuo>eozocwquu.ma .mflm 67 18W 161- L—(unOH/’Od 10W") AllOO'lElA/l. (1/mMI 1/[SUBSTRATEI 68 .xun>nuoe omemh< o>nunmcomcnucneneso ocu manna >un>nuoe omemh< Hench on» me eocnmov we: xun>nuoe omemh< +x\+ez .nzocm one euee ozn now mocnn unm monescm numeog .mH onSMnm now ecomon onu an wonnnomoe ono3 mconnnecoo nozno .woms we: Ht connenemonm umocm 0mm .xna :onpensocn enevceum oau on :Onuneee an :nepeso 25 H uocneucoo xne sownepsocH .mn onDMnm now ecomoa osp an me one mHonExm .monennEoE nmosm umm sonm omemh< +x\+ez ”uon xnsmuno>eo3ocnquu.oH .wnm 69 l—(unOH/Vocs 1owfi) AllOO'IBA/l (1/mM) 1/[SUBSTRATEI 70 Ouabain-insensitive ATPase was also stimulated by the 30-50% cut (see Figures 17 and 18). Brain microsomes were assayed in the presence of the used dialysis buffer and/or the 30-50% cut. The Lineweaver-Burk plot is shown in Figure 19. The 30-50% cut generally stimulated the activity of total ATPase of mouse brain microsomes. All lines drawn on the Lineweaver-Burk plots were by least- squares fits of the data. Rabbit Trachea Ciliary Response to Sera The rabbit trachea ciliary assay was performed as indicated in the Methods section. Six C/P serum samples and 6 control serum samples were tested. Explants from the same rabbit were used. The results were averaged and are shown in Figure 20, which displays the percent of maximum response versus time of exposure to the serum sample. The figure shows that the percent of maximum response of rabbit tracheal ciliated epithelium to C/F sera was greater than the percent of maximum response to control sera. The time required to reach one-half maximal response was less with C/F sera than with control sera. Movement of cilia was significantly different at 20 minutes (p < 0.1), 25 minutes (p < 0.3), and 30 minutes (p < 0.1). Secretion of debris was significantly different at 15 minutes (p < 0.1), 25 minutes (p < 0.01), and 30 minutes (p < 0.05). The test used was a two-tailed Student's t-test without assumption of equal variances. 71 .vcomoH mH onSMHm ox» an me one mnennonee one mvonuoe nocno .onswnm ens» :o wonuoam cacao one mocnn paw monescm -umeoq .wom: me: we acnuenemonm umozw 0mm .xne conuensocn unevceum on» on vowee we: 5:0nnenucoo Icoo Hecnm ZS Hy eneneso .mH onsmwm now vaowon ogn an me one mHonsxm .uso wom-om mo m:0nuenn Icoocoo wcnxne> can: mocennaoa amonm 0mm Eonm omemp< o>nunmcomcnucnepeoo "pang xnsmuno>eo3ocnquu.nfi .mnm 72 l—(HnOH/‘bd 1owfi) Alison/m (mMI‘1 1/ISUBSTFIATEI 73 .mH onzmnm nom ecomoa ocn an me one meocpoa one mAennoueE nosno .nzocm one mocnfi unm monencm-umeo4 .voms we: we acnuenemonm amonw one .3 com AI .. I .8 A: 03 All .8 A: 02 AIII .3 A: om A ..... .3 ”wom-om o: .¢IIIII .ov ”one onpexm .EOMnenncoo Icoo Hecnm 25 A on xfie coaueDSUEn eneecenm ocu on wovwe we; aneneso .uso womuom mo m¢0nuenu :coocoo mcwxne> ann3 mocennsoe umocm 0mm Eonm omeme< o>nnnmcomnfiunweneso "noam anmuno>eo3ocnguu.wH .wwm 74 l—(HnOH/Vocl 10w”) AllOO‘IaA/L I1/mMI 1/[SUBSTRATEI 7S .euee ocp mo unm monescmuumeoa on we“ -vnoooe :3env ono: mocng .mopscns om on mooseon we: oEnu :Onuenzocn meow: ono: moeomonone :Henn omsoa mo mcfiouonm m: mmv A: om unmooxo pom: ono: ma onSMnm now me meanunvcoo xemme oEem on» one m:0nnenncoo:oo use womuom oeem och .vcomoH mA onswnm onu ca me one mHonexm .uso womuom mo m:0nuenu:oo:oo mcnxne> can: moEOmonone :nena omsoe Eonm omemha Heuou "uoam xnsmuno>eo3ocnqun.mH .wnm 76 22:5; .m...ov wnevceum use momeno>e och .omcommon AeEner mo ecoA no .om .o wovenm ono: momcoamom .mHe>nou:w onscna m ne cone=He>o ono: EdnAocuMmo wouenfifio on» we momcommon on» one .voxemme ono3 enom m\u 0 one Honncoo Ham .cofiumfinomoe :Onuoom meonpoz onu on mcflenoooe voenomnom we: xemme eocoenu unnnen och .oEnn can: enom on asnaonuflmo eonoenn unnnen mo momcommon ofipocnxmxv one xnouonoom mo cenmmonmxm-u.om .mfim 78 _ — _ — mmmm 1 30 25 mmZOmwmm EDS—33:2 no PZwomwm $qu \>U em use Donn an mneo; an on a: non vonensucn osmmnn one .mmH ennvoz no enom Aonn -coo no n\u A>\>V emu ece ocneemooenm-An-zmv no: mA nun: mmA Edneoz smonm on wannnommcenu onomon exee N now onanHSU an bonnennnea me: EdnHonnnmm .nceAmxo ocn mo ouecomoeo: onu an anononm me A on wounAeEno: ono3 .2.m.u .mmH ennvoz no AA>\>V emm any enom nonneoo no n\u non: eoneonn ono: Ednfiosnnmo wouenfino eonoenu unnnen mo mouecomoeoz one .Hennoues oAnennmnoonm <0h oucn ocneemoosfim o>nnoe0nuen mo osnn can: connenomnoocm-s.A~ .wnm 81 5r- .tha Game: 323.2 3555.2 SeEEGmE «on E E< 2017:... TIME (HOURS) 82 .mnso: oH mo eeoumcn mnso: cm on a: now wonensocn onoz mofimsem one ocone oon seneoz no oeoom:n mun A>\>o emu can: woneonn we: moameem we now one Hues» umooxo .mnneuoe newcoEnnomxo now AN on:Mnm oom .oneco -moeo: mo :nononm me A on wounaeeno: onoz xnn>nuoe -Onven mo m:0nnenomnoocn .mom no enom Honucoo no m\u gun: eoneonn ono: Ednnocunmo eonoenu pnnnen mo mouecowoso: .Hennoues oAneunmnoonm <09 ounn onneemoozflm o>nnoeonuen mo osnu can: connenomnoocnu-.m~ .mnm 83 _° N _o ._ _.N ,_ .J o I '2 oo 8&0 ILUU 40:] “Q 1 l 1 L 1 1 o O o O o o D C O o N o a o 8 § 1- ‘- (anssu fiwl'wd'o) wuualvw 318Vl|dl038d V01. OlNl Nouvuowoom TIME (HOURS) DISCUSSION The basic defect in C/F is not known at this time, and researchers have little idea of the nature of the basic defect due to a lack of understanding of the processes differing in C/F and control individuals (i.e., the pathology of C/F is only understood at the clinical level). However, one generalization can be made about the pathogenesis in C/F: some of the functional activities of the cell coat and/or the cell membrane of C/F tissue or C/F treated normal tissue are altered when compared to controls. The research presented here has been on three materials on or in contact with the cell coat and cell membrane: CBLA, ATPase, and the tracheal epithelium ciliary border. Each topic will be discussed below. CBLA: Introduction CBLA was implicated in C/F as the basic defect and was thought to be deficient in C/F sera. Lieberman (1975) and Corbin et al. (1976) showed that CBLA was not significantly lower in C/F; moreover, in certain C/F patient groups it was elevated. The question of the existence of an isozyme of CBLA in serum has been raised and will be discussed later. The significance of an isozyme of CBLA to C/F research may not be great since Bowman et a1. (1975) presented evidence that the ciliary inhibitor does not cross-react with mono- specific anti-C3a. (Conover et al. (1974) had suggested that C3a 84 85 was the ciliary inhibitor, which they hypothesized to be in excess in C/F because anaphylatoxin inhibitor (CBLA) failed to inactivate it.) CBLA Assay and Purification The CBLA assay was shown to be valid by its meeting the two criteria that: product formed (hippuric acid) is released linearly with time; and that enzyme velocity is linear with enzyme concentration. See Figures 1 and 2, respectively, which show that the assay met the two above criteria. Cohn Fraction IV-l was used as a source from which CBLA was purified. At pH 8.0 in 50 mM potassium phosphate buffer CBLA was capable of binding to TEAE cellulose; but when the ionic strength was raised using a linear 0 to 0.3 M NaCl gradient, CBLA was eluted from the TEAE cellulose. As seen in Figure 3, the activity was eluted in two peaks. The elution of activity in two peaks suggested that either isozymes of CBLA exist or that part of the activity was bound to a second protein while the remainder of the activity did not bind to that second protein, giving two peaks of activity eluting at different ionic strengths. The CBLA peak that eluted first coincided with the elution of the peak of protein concentration. This fraction of activity was not further purified. However, if it were purified, it would be interesting to compare it with the subunit structure of the second activity peak on denaturation with urea or guanidinium HCl and analytical centrifugation. The first activity peak was not included in Table 2, the Enzyme Purification Summary Sheet, and that loss is reflected in the drop in yields from 85.2% to 21.6% recovery between Steps 2 and 3 86 (i.e., before application to the 5 x 80 cm TEAE column to after its elution). The fractions making up the second activity peak were pooled, concentrated, and applied to Sephadex G-200. The elution profile (Figure 4) showed the separation of CBLA from a higher molecular weight protein peak. The fractions containing activity that were pooled were selected in a narrow range. This procedure sacri- ficed activity, as indicated by the decrease in yield from 21.6% to 4.46%, but it also removed a large amount of extraneous protein. 0n the second G-200 column little activity was lost: the yield was reduced from 4.46% to 3.99%, but total protein was reduced by about a factor of 5. The fold purification of all the procedures was 86.5. This value compares with a 14% yield with a 252 fold purification from serum by Erdos et al. (1967) and with the purification of Bokisch and Mfiller-Eberhard (1970) of anaphylatoxin inhibitor from pseudo- globulin who obtained a preparation of 280 uMol hippuric acid released per minute per mg protein with a yield of 6%. The CBLA purified as indicated in the Methods and Results sections was undoubtedly not purified to homogeneity. If a very high specific activity preparation were needed, additional methods to be used in purification would include affinity chromatography. The method of Sokolovsky (1974) could be used to prepare a suitable column. He bound EACA to CNBr-activated Sepharose and, using N, N' dicyclohexylcarbodiimide, linked the EACA spacer to arginine. A precaution which could not be taken during purification was to run all purification steps at 4°C instead of 23°C. While this purification was being done, there was no access to a cold room. 87 Since columns usually took 24 or more hours to develop, the maintenance of the higher temperature probably contributed to a substantial loss in yield. If columns were run at the lower temperature, the peptidase activity would have been about 4-fold lower (assuming Q10 = 2.0), and considerably less enzyme would have been self-destructed. Further recommendations for CBLA purification include: in place of the second G-200 column, run a column of Sepharose 68; run a 5 to 20% (W/V) sucrose gradient centrifugation to separate the CBLA based on sedimentation coefficient as opposed to separation based on charge properties and Stoke's radius as in previous steps; and run prepara- tive isoelectric focusing. Phosphate Determination The technique used for phosphate determination described in the Methods section was a modification of the techniques of Fisk and Subbarow (1925), Ames (1966), and Drewes (1972). All of the above techniques required incubation periods before the optical density was measured. The modification of the above techniques, as presented in the Methods section, eliminated the incubation period. The method was nearly as sensitive as the method of Ames who reported a molar absorbance of 2600 A/M/cm at 820 nm, and was more sensitive than the method of Drewes who reported a molar absorbance of 732 A/M/cm. The color stability of the technique was reported in the Results section, and as indicated in Figures 7 and 8, the color diminished slowly and the rate of loss was linear with the concen- tration of phosphate. However, the fastest rate of color loss was -0.00122 A/min. Consequently, optical densities were measures within 88 2 to 3 minutes after the addition of ethanolamine so that the amount of color change was no more than the size of the measurement reli- ability of the spectrophotometer. The wavelength scan results shown in Figure 9 indicated that the absorbance peak corresponding to the reduced phosphomolybdate began at 500 nm and increased through the end of the scan at 750 nm. A point of inflection in the curve was located at 550 nm, and the shape of the curve indicated that the wavelength of the maximum was roughly 800 to 850 nm. This estimate corresponded to the value obtained by Ames of 820 nm. The optical density routinely was measured at 580 nm due to the inaccessibility of a spectrophotometer capable of reading at a higher wavelength. The suitability of reading at that wavelength, given that no higher wavelength could be used, is justified, inasmuch as the value at 580 nm was about 67% of the maximum value at 750 nm and about 80% of the molar absorbance of Ames. It also was shown that any wavelength below 550 nm was unsuitable. Below 550 nm results were inconsistent due to the low molar absorbance of reduced phos- phomolybdate at those wavelengths and partly due to the rise in optical density of the blank. Figure 10 illustrated the magnitude of the increase in optical density with decreasing wavelength when no phosphate was present in the sample. The reference to which the sample was compared contained only distilled water. ATPase Assay Validation The ATPase assay as described in the Methods section was shown to meet the two criteria for assay validation: first, product 89 formed (Pi) was shown to be produced linearly with time (up to 90 minutes), and second, the rate of Pi formation was shown to increase linearly with enzyme concentration. See Figures 11 and 12, respectively. The Effect of Serum Proteins on ATPase Activities As seen in Figures l3, l4, and 15 the addition of 30-50% cut of pooled human serum caused an increase in total ATPase activity in human RBC ghost membranes. This increase in activity was also shown with Na+/l(+ ATPase (Figure 16) and ouabain-insensitive ATPase (Figures 17 and 18) of RBC ghost membranes and total ATPase of mouse brain microsomes (Figure 19). The stimulation of RBC ATPase activity by immunoglobulins was reported by Bader (1971) as preliminary data, and Schmoyer and Baglia (1974) reported that ATPase activity from RBC ghosts was increased by the addition of dialyzed unused media supplemented with FCS added to the assay mix. Schmoyer and Baglia used 0.02 M TBS pH 7.5 as a control against which ATPase treated with media was compared. The results reported on the Lineweaver-Burk plots indicated that there was activation by nondialyzable material and that the amount of activation was dependent on the concentration of the 30-50% cut added. In addition, most of the least-squares fit lines ran roughly parallel to each other. For these reasons, the kinetics did not rule out the possibilities that the 30-50% cut affected the ATP- enzyme complex, that the 30-50% cut supplied one of the substrates, or that the 30-50% cut acted to aid the enzyme in binding one of the substrates. Alternatively, the kinetics did not rule out the possibility that the 30-50% cut contained a molecule (a subunit of 90 the enzymes) that was bound to the ATPases in_!i££2_but that was lost during the preparation of the enzymes. Future tests of the effect of 30-50% cut on ATPases include: the effect of heat-inactivated 30-50% cut, the effect of 30-50% cut which was heavily dialyzed against chelating agents, the effect of DEAE and CM cellulose fractionated 30-50% cut, and the effect of 30-50% cut fractionated by gel filtration. Further kinetic experi- ments using wider ranges of 30-50% cut concentrations are also planned. The continued investigation of the activation of RBC ghost and brain ATPases will hopefully allow the determination of how this apparently normal physiological activation occurs. Knowledge of the mechanism and functional components involved in thos process will aid in the interpretation of the results of Schmoyer and Baglia (1974), Cole and Dirks (1972), and Cole and Sella (1975). They showed that C/F serum, saliva, and used medium from fibroblasts contain materials which inhibit ATPases or activate ATPases less than the corresponding control material. It was hoped that the data reported here would serve as a baseline from which the normal physiological and biochemical situation could be understood; and having evaluated the normal condition, a comparison of the effect of C/F and control materials on ATPases with an interpretation of the differences could be made. Rabbit Trachea Ciliary Bioassay The rabbit trachea ciliary bioassay has provided a great deal of information on the differences between C/F, heterozygote, and 91 control materials (either serum or used medium from cultured cells). See Conover et al. (1973a, 1974), Yeates et al. (1976), and Spock et al. (1967). However, Wood and di Sant'Agnese (1973) pointed out that the bioassay was unreliable as a diagnostic test in differenti- ating C/F homozygote and heterozygote sera from control sera. Results from this laboratory indicate that one cause for the unreliability of the bioassay was the need for an observer to evaluate the state of the tissue. A second drawback was the variability of responses to the same test sample from rabbit to rabbit and to a lesser extent from explant to explant. Other assay deficiencies were that the threshold level for response was high (preventing the assay of diluted material), and the magnitude of response was not great. Moreover, only a very few samples could be observed during one run which took 30 to 35 minutes; consequently, the bioassay was tiresome and difficult to perform. Despite the multitude of disadvantages, data were obtained following the procedure described in the Methods section. As shown in the Results section the magnitude of the secretory response of the tissue to C/F sera was significantly greater beyond 20 minutes exposure to C/F serum, as compared to control sera. Other observers have reported a wide variety of incubation times prior to the initiation of a CD+ response to C/F serum. Conover et al. (1973a) reported CD+ response times of 3 to 6 minutes, Sturgess (personal communication) reported 10 to 15 minutes, and Cheung and Jahn (1976b) reported 1 to 2 hours. These variations in 92 response time were undoubtedly dependent on technique and animal variation. Future Experiments Using_the Ciliary Bioassay One problem in quantifying the ciliary bioassay is that the mechanism of the secretory and dyskinetic response of rabbit tracheal epithelium has not been characterized. A proposal to investigate the mode of the epithelial cells' response is to examine the necessity or lack of necessity for microfilament and microtubule fUnction in put-um .‘h , : H the ciliated cells in order to elicit a CD+ response on treatment with C/F serum. Another subcellular requisite to elicit a CD+ response on treatment with C/F is hypothesized to be ATP. The pre- treatment of explants with uncouplers would lead to intracellular ATP depletion and might eliminate CD+ response capability. Another hypothesis is that C/F serum causes the lysis and death of the ciliated epithelial cells; this hypothesis is being tested by using time-lapse microcinematography of the epithelium in the presence of C/F or control serum. Lysis and death of the ciliated cells will be detected by vital stains. Suitable stains include trypan blue, eosin Y, and erythrosin B, all of which are excluded by viable cells, and accepted by lysed cells. Incorporation Experiments The incorporation of (SH-l)-glucosamine into rabbit trachea epithelium treated with C/F and control sera was tested as a possible alternative to the ciliary bioassay. The results indicated that there were no significant differences in the amounts of incorporation 93 of tritiated glucosamine into the TCA precipitable material in the homogenized explants. Although this particular method did not provide a quantified assay to replace the ciliary bioassay, it did indicate that C/F serum had no toxic or stimulatory effect on the incorporation of glucosamine into the TCA precipitable material of the explant. However, the explant contained a mixture of cell types, and it could be argued that only the ciliated epithelial cells were affected, while none of the other cell types were affected, masking the alter- ation in epithelial cell function by the C/F serum. It should be pointed out that the experiment described here used a crude and preliminary method. Effects possibly contributing to the lack of sensitivity of this technique are: (1) entry of glucosamine into the cell could be affected by C/F and control sera (2) C/F and control sera could affect the pool size of glucosamine in the ciliated cells (3) the possibility exists that only mucopolysaccharide, glycosphingolipid, or glycoprotein synthesis alone is affected by C/F or control serum, and the other two synthesis rates are similar (4) C/F sera might increase synthesis of a particular glucosamine incorporating product and decrease synthesis of other pro- ducts, giving roughly equal amounts of glucosamine incor- poration into TCA precipitable material (5) the steady state amount of incorporated glucosamine with C/F and control sera treatment might increase at the same rate; 94 while the rate of secretion of incorporated glucosamine might be very different (6) more than one of the above effects may occur when ciliated epithelium is treated with C/F serum. Further experiments include the separation of glycosphingo- lipids from glycoproteins and acid mucopolysaccharides of the ciliated epithelium which was incubated with radioactive glucosamine and C/F or control sera. The lipid material (containing the glycosphingolipids) will be extracted from the homogenized material with chloroform: methanol (2:1). The examination for possible differences in the levels of glycosphingolipids between C/F and control sera treatment can be checked in this manner. Changes in the level of synthesis of particular glycoproteins and mucins labelled with radioactive glucosamine can be monitored by polyacrylamide gel electrophoresis of the homogenate of the ciliated epithelium treated with C/F or control serum and radioactive glucosamine. The incorporation into specific fractions could be tested by slicing the gels into thin discs and counting the slices in a scintillation spectrophotometer. Ongoing experimentation using incorporation of tritiated glucosamine as an indicator of glycoprotein and mucin synthesis include: (1) A test of the levels of nondialyzable radioactivity in the medium of rabbit trachea epithelium incubated with C/F, control, and FCS and tritiated glucosamine. This experiment Pm ..» _ (2) (3) 95 will indicate the effect of the sera on the production of secreted soluble, nondialyzable glycoproteins and/or mucins. A pulse and chase experiment--pulse label the ciliated epithelium with tritiated glucosamine and chase with non- radioactive glucosamine with C/F, control, or FCS. Tissue explants are examined in histologic sections via autoradiography. The purpose of the experiment is to examine for heightened or " decreased synthesis of glycoproteins caused by the C/F sera. Injection experiment--young mice are injected with tritiated glucosamine and later with C/F or control sera subcutaneously in the neck region. Thirty minutes later the mice are sacrificed. 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