m VETRO PRODUCTION DISC GEL ELECTROPHORETIC ISOLATION AND PURJHCATLON OF RAT PROLACTLN Géssefiation for the Begree of Ph. D. MESH! GM STATE UNIVERSHY KE‘LRE' H H KGRTREGHT 1 9 7 3 m, .5! 'fl I IJIJ:;;:“ {Y ‘ ‘ Michigan State University This is to certify that the .._n'...$ o‘- .‘ o 5 ' . ‘_ p thesis entitled In Vitro Production, Disc Gel Electrophoretic Isolation and Purification of Rat Prolactin ' presented by : . Kenneth H. Kortright has been accepted towards fulfillment of the requirements for Ph .D . degree in PhysiologL E 5/7: ‘ é Major professor Date November 9, 1973 0-7839 ' i. I; amomc av. ‘5 HUAG & SUNS' BIIIIK BINDERY INC. LIBRARY BINDERS I I’lflfl'fll! Ilcllflll ABSTRACT IN_VITRO PRODUCTION, DISC GEL ELECTROPHORETIC ISOLATION AND PURIFICATION OF RAT PROLACTIN By Kenneth H. Kortright An efficient method for the isolation and purification of biologiw cally and immunologically potent rat prolactin in high yield is herein reported. Adenohypophyses from estradiol benzoateapretreated female SpragueaDawley rats were organ cultured for periods of up to eight days. Medium was harvested from these cultures every two days until their termination. The individual batches of medium were separately pooled, centrifuged, and concentrated. Volume reduction was accomplished on an Amicon Diaflo apparatus at 20 PSI N2 (nitrogen) pressure. Prolactin was isolated from these media by preparative polyacrylamide disc gel electrophoresis. The electrOphoretic column employed a 2.5% polyacrylw amide stacking gel, 7.5% polyacrylamide resolving gel, with a modified Tris-HCl, Trisuglycine discontinuous buffer system. Column fractions exhibiting absorption peaks at 280 mu were pooled and dialyzed for 48 hr. The dialyzed fractions were concentrated as before but at 55 PSI N2 pressure. Following concentration these samples werelyophilized. The recovery of radioimmunoassayable prolactin from the starting medium was 67.6 :_3.3% representing a yield of 42.3 :_5.8 mg/gm of wet weight of pituitary tissue. The mean biological potency of 3 isolated batches was Kenneth H° Kortright 29.0 :_.95 IU/mg. The efficiency of this system represents a signifim cant increase in yield of rat prolactin equivalent in potency to the best rat lactogenic hormones reported thus far in the literature. These isolated fractions were compared immunologically and biologically with all other rat prolactins presently available. The gain in effi« ciency of this system was enhanced by using pituitaries from female rats pretreated with estrogen. Adenohypophyses from young female rats that had been pretreated jn_!jvg_with estrogen released 41% more pro« lactin than non-treated females. Moreover. pituitaries from old female rats pretreated with estrogen inpvjyg_released 105% more prolactin than young pretreated females, and 69% more than old estrogenmpretreated males. Combining these effects with a starting medium of high prolactin— low total protein ratio organ culture medium, an efficient purification technique, and a high quality and quantity yield, provides an economical method for rat prolactin isolation and purification. The statistical computations of pigeon cropasac responses in the bioassays were treated differently than thus far reported. 'A linear rem gression equation was developed and precise confidence intervals were set on both standard dosewresponses and predictions of unknowns. This method permitted as competent a means as available to account for hetero- geneous variance in bioassay responses. Covariate and weighted regression analyses were applied as well but found to be unsatisfactory due to extreme variances encountered in individual pigeon responses. Thedegree of these variances were correlated with the season of year in which the assays were performed. I VITRO PRODUCTION, DISC GEL ELECTROPHORETIC ISOLATION AND PURIFICATION OF RAT PROLACTIN By . a "“ Kenneth HIIKortright A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1973 ,‘J _ Ch ACKNOWLEDGEMENTS A statement praising all of those generous and empathetic people who contributed to the development of this chronicle on a biological research problem might better be achieved by a twentyefour hour eulogy. However, in view of tradition and economy, I will penuriously bestow my appreciation to those most deserving. My gratitude is extended to Dr. Joseph Meites for providing the laboratory space and facilities necessary to work on this problem. Members of his laboratory, past and present, who aided in developing the data herein reported were Dr. Steven w. Clark, Gregory P. Mueller. and Mary K. (Spindler) Vomachka. The Biochemistry Department's Dr. James L. Fairley, his student Richard Jagger, and the electronics shops' Ben Stutsman were all helpful in their offers of advice and encouragement. Professor John L. Gill unselfishly interrupted many home and aca« demic commitments to offer aid, advice, and encouragement. in fording the rapids of statistical analysis of the data. To Dr. Gill I offer my heartfelt gratitude. To Dr. Robert K. Ringer, who pops chicken pituitaries from their jg_situ confines like blanching almonds, I can only say thank you from from the bottom of my heart. Job placement, consultation, and encouragement was generously and sincerely offered by a grand renal physiologist. Thank you very much Dr. Collings. Dr. William L. Frantz contributed greatly to my understanding and applications of the prolactin physiology in this work. Moreover, his wise and considerate advice (personal.academic, and professional) were greatly appreciated. Thanks are also given to Dr. H. Allen Tucker for reviewing the thesis in Dr. Hafs absence. Last, but not least, of my guidance committee is the seasoned and crusty philosopher Dr. E. Paul Reineke. Words cannot express feeling for a man you would be proud to call Dad. His pursuance of statistical analyses of thesis data is sometimes extreme, but that is the Sam Brody in his blood. What thanks could mean. Paul, words will never say. There are two secretaries who have made this all possible. To them. a note in these acknowledgements should be selfwexplanatory. My warmest gratitude to both of you—~Amylou Davis and Nancy Turner. Thanks! I also have a son, Kristopher Scott, whom I will long to have at my side one day. His existence continues to drive my energies towards being the best man, scientist, and father, I can possibly be. There is another person that deserves special thanks for her warm and sincere encouragement, during periods when all life seemed cold. To Mrs. Leona (Leigh) A. Dunn. thank you for being you. TABLE OF LIST OF FIGURES O O O O O O O 0 CONTENTS INTRODUCTION. . . . . . . . . . Prolactin History. . . . . Prolactin Bioassay History Other Assays . . . . . . . Prolactin Biosynthesis . . Method of Isolation. . . . MATERIALS AND METHODS . . . . . Experimental Animals . . . Organ Culture Procedure. . Concentration of Medium. . Preparative Disc Gel ElectrOphoresis Processing Column Eluants. . . . . . Concentration of Dialyzed Fractions. Analytical Techniques. . . . . . . Radioimmunoassay Procedure . . . . Lowry Protein Test . . . . . . . . Iodination of Rat Prolactin. . . . Analytical Polyacrylamide Disc Statistical Analyses . . . Radioimmunoassay Results . RESULTS 0 O O O O O O O O 0 O 0 Organ Cultures . . . . . . Medium Processing. . . . . Pigeon Crop-Sac Bioassay . DISCUSSION. . . . . . . . . . . SUMMARY AND CONCLUSIONS . . . . . APPENDICES Gel A. Terminology for the Biological iv 0 O O O O O O O O O O O O O O O 0 O O O 0 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 O O O Electropo esi o 0 "so 0 o o o o o o o o o o in. o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o O 0 go 0 O O O O C O O 0 O O O 0 Actions of Prolactin. 0 0 O 0 O 0 O O O 0 O O O O O O O 0 0 0 O O O O O O O O O Page vii £005de 53 APPENDICES-~Continued AA. B. Endocrinology Study Section. . . . . . . . . . . . . . . Prolactin Released by Adenohypophyses from Pretreated and Non-pretreated Female Rats . . . . . . . . . . . . . Electrophoresis Reagents . . . . . . . . . . . . . . . . Effects of Age and Sex on Prolactin Release 12 vitro by Rat Adenohypophyses . . . . . . . . . . . . . . . . . Prolactin and Protein Production in_vitro. . . . . . . . Concentration of Organ Culture Medium Under Different conditions 0 O O O O O O I O O O O 0 I O 0 O O O 0 0 0 0 Absorbance (Abs.) for the Protein Elution Profile of Preparative Fraction KKmRPul . . . . . . . . . . . . . . Mean Absorbance (Abs.) of the Preparative Column Eluants Preparation Column Protein and Prolactin Elution Pro» fiIeSoooooo0.900000000000000099 Prolactin Losses During Each Step of the Isolation syStem O O O O O I O O O O O O O O O O O O O C 9 O O 9 0 Summary of the Dose Response Curve Responses for the PCS Bioassayooooooo0.000000000090000 Linear Regression Analysis of the Pigeon Croprac Bioa assay DoseuResponse Curves . . . . . . . . . . . . . . . Dose ReSponse Data for the PCS Bioassays Standards . . . Body Heights of Assay Birds Employed in Developing the Dose Response Curves . . . . . . . . . . . . . . . . . . Summary Table of the Bioassays of Isolated Column Fraca tions and Prolactin Standards. . . . . . . . . . . . . . Biological Potency Evaluations of the Rat Prolactins in IU/mQO O O O C O O 0 O O 0 O O O O O O O O O 9 O 0 9 9 O Bioassay Responses for the Analysis of Isolated Column Fractions and Prolactin Standards. . . . . . . . . . . . Page 54 56 57 58 59 61 62 63 64 66 67 68 69 70 71 72 73 APPENDICESwnContinued Z. Dose Response Curve Results of Column Fractions and Prolactin Standards in the Radioimmunoassay (RIA). . . . Fifty Per Cent Binding Values of Radioimmunoassay analy= sis of Column Fractions and Prolactin Standards. . . . . Summary Table of the Analyses of Biological and Immunow logical ActiVity of Isolated Column Fractions. . . . . . Summary Table of the Analyses of Biological and Immunoa logical Activity of Isolated Column Fractions. . . . . . CompariSon of Biological and Immunological Activities of Column Fractions with Available Rat Prolactins . . . . . Comparison of Biological and Immunological Activities of Column Fractions with Available Rat Prolactins . . . . . Relative Comparisons of the Isolated Column Fractions and Rat Prolactin Standards. . . . . . . . . . . . . . . Relative Comparisons of the Isolated Column Fractions and Rat Prolactin Standards. . . . . . . . . . . . . . . Summary of Biological and Immunological Activities of Column Fractions with Available Rat Prolactins . . . . . Summary of Relative Comparisons of the Isolated Column Fractions and Rat Prolactin Standards. . . . . . . . . . REFERENCES. 0 0 O O O O O O O O 0 O O O O O O O I O O 0 O O O O 0 vi Page 75 76 77 78 79 80 8T 82 83 84 85 LIST OF FIGURES FIGURE Page l. Prolactin Isolation Flow Chart. . . . . . . . . . . . . . . 23 2. Cumulative yield of prolactin by controls and estrogen treated rats 0 O O O O O O O O O O O O O O O I O O 0 O O O O 24 3. Effects of age and sex on prolactin release j§_vitro. . . . 26 4. Prolactin and protein release in vitro. . . . . . . . . . . 28 5. Mean recovery of prolactin at six stages of prolactin isoa lation. C O O O O O I O O O O 0 O O O O O O O O O O O 0 0 0 32 6. Prolactin and protein recovery from 5 batches of culture medium concentrate. . . . . . . . . . . . . . . . . . . . . 33 7. Preparative column protein elution profile. . . . . . . . . 35 8. Preparative protein elution profile, mean for five runs . . 36 9. Preparative column protein and prolactin elution profiles . 38 l0. Pigeon cropusac bioassay standard curves for three experiw ments 0 O O O O O O O O O O O O O O O o O O O O O O O O O O 40 vii INTRODUCTION Prolactin History The seemingly ubiquitous nature of the actions of prolactin. which hails by a variety of aliases (prolactin, galactin, galactopoietic hora mone. mammotropin, mammotropic or mammogenic hormone, lactogen, lactou genic hormone, luteotrophic hormone, luteotropin, or paralactin) has recently been reviewed by Bern and Nicoll (T968) and Nicoll and Bern (1972). "Prolactin" (PRL) is the name coined for the first anterior pituitary (AP) protein hormone to be extracted in partially pure form (Riddle et al., 1932a & b, 1933). This active principal has become most prominent in worldwide research interest owing to its multiplicity of actions in numerous vertebrate species (Nicoll and Bern, 1972). Prolactin's role in mammary gland development. function, and tumourin genesis has largely contributed to the interest surrounding this hormone (Meites et al., l972a, b, & c). To characterize the rapid advancement of the study of PRL, the amino acid analysis of ovine PRL wascompleted very early by C. H. Li (l949) and sequenced by the same investigator (Li, 1969, 1972), as well as others (Seavey and Lewis, 1971). Recent technological advancements have permitted more efficient isolation, with less molecular damage, of adenohypophyseal protein hormones.' These methods will be reviewed under PMethods of Isolation“. In order to appreciate the approaches utilized in these isolation studies and their findings, a resume of the history and physiological roles of PRL is essential. Interpretation of some of these studies may be enhanced by Appendix A, which tabulates the terminology employed in describing the biological actions of PRL. Prolactin Bioassay History The existence of PRL, initially called "galactin" for its galactou poietic activity in rabbits (Turner and Gardner, 1931), was demonstrated by Stricker and GrUter (1928, 1929). These workers discovered that some anterior pituitary principal was responsible for the initiation of lactation in previously developed mammary glands of the rabbit, bitch, hog. and cow. It was soon after concluded that this lactational response was not due to the gonadwstimulating hormones (GrUter and Stricker, 1929; and Corner, 1930). Using extracts similar to those of Corner (1930), Turner and Gardner (1931) brought spayed virgin female rabbits into com— plete mamnary development. These findings were confirmed in ovariectomz- ized rabbits by Asdell (1931), and Nelson and Pfiffner (1931). Riddle and Braucher (1931) demonstrated that this same principal could stimulate mucosal epithelial proliferation ("crop milk", Beams and Meyer, 1931) in doves and pigeons. Riddle and Braucher (1931) were also the first investiw gators advocating the use of pigeon cropwsac proliferation as an assay procedure for the lactogenic principal. Using beef, sheep, and hog pituiw taries the lactogenic hormone was extracted, partially purified, named "prolactin", and utilized in developing a preliminary crop-sac bioassay in pigeons (Riddle et al., 1932a & b, 1933). Other reports confirming the crop-sac, "crop milk" production, assay method soon followed: Lyons and Catchpole (1933a & b), Lyons and Page (1935), McQueeanilliams (1935), McShan and Turner (1936). Refinements of these methods, reporting the later accepted bioassay of PRL, were given by Lyons (1937) and Reece and Turner (1937). The preferred method was based upon intradermal injections of 0.1 ml volumes of test materials into the feather follicles over the cr0p of fledglings. Following a four day injection regime, using both ventrolateral sides of the crop-sac as test sites, the pigeons were killed. The cropasac was then removed, cleared of excess fat and muscle tissue, held to the light and subjectively rated upon the area of mucosa epithelial proliferation using a rating index of one to four. The credit for developing this "microumethod" of the pigeon crapusac assay (PCSAm) was given to Lyons (1937). More recent developments of this technique are given in Tanabe et a1. (1954), Grosvenor and Turner (1958), and Kanematsu and Sawyer (1963). The presently accepted and most quantitative version of the cr0pasac bioassay, micrommethod, is that of Nicoll (1967). Nicoll‘s bioassay method was used in this study. Systemic injection regimes, although less sensitive an assay than the intradermal method, were reported by Lyons (1937) as the “macro-method“ and later modified and refined by Bates et a1. (1963), and Nicoll (1969). Other Assays A proliferative mitotic activity in the crapwsac had also been demonstrated in response to PRL (Bergman et al., 1940; Dumant, 1965) which coincided with nucleic acid synthesis (Brown et al., 1951; McShan et al., 1950; Bern and Nicoll, 1968). Using this knowledge Damm et al. (1961) developed a PRL assay in pigeons on the basis of 32P uptake by the crop-sac epithelium. Ben-David (1967) developed a similar assay on the basis of 3H-methyl-thymidine uptake by cropmsac epithelium. More recently, double antibody assays called radioimmunoassays (RIA), emu 131I-or 125I-labeled standards, have been developed. Arai and ploying Lee (1967) and Bryant and Greenwood (1968) developed the ovine prolactin RIA; Bryant and Greenwood also measured bovine PRL with the ovine PRL RIA. Kwa and Verhofstad (1967) and Niswender et a1. (1969) developed the rat prolactin RIA, and Bryant and Greenwood (1972) developed the human prolactin RIA. The latest of these assays, which is a measure of both immunological and biological activity of PRL, is the radioreceptor assay (Frantz and Turkington, 1972; Shiu et al., 1973). These receptor assays are capable of measuring multispecies prolactins. The lactogenic or mammotropic actions attributed to prolactin were based upon the ability of this principal to initiate lactation in developed mammary glands of many species. PRL also initiated sequential development of the lobuloalveolar and ductal components of the mammary tree as demonstrated by Corner (1930), Turner and Gardner (1931), Asdell (1931), Nelson and Pfiffner (1931), Gardner and Turner (1933), Lyons and Catchpole (1933a & b), Lyons (1937, 1941, 1942), Bergman and Turner (1940), Folley and Young (1941),and Gardner and White (1941), as reviewed by Lyons (1941, 1942) and Folley (1952). A review of the methods of assaying for mammotropic, lactogenic, and crop—sac stimulating activities of prolactin is given in Bergman et a1. (1940) and Lyons (1941). The latest assay developments for lactogenic activity involve the synthesis of casein by mammary tissues in vitro (Turkington et al., 1965; Turkington and Topper, 1966; Turkington et al., 1967; Beitz et al., 1969; R. L.Ceriani, 1969; Frantz and Kleinburg, 1970; Frantz et al., 1972; and Turkington, 1972). The "luteotropic hormone" title attached to PRL derives its name from the ability of this hormone to produce intense luteinization of the ovaries in virgin rats while concomitantly producing full development of the mammary glands (Selye et al., 1933a & b, as reviewed by Riddle et al., 1933). Moreover, Evans and Long (1922) had much earlier indi= cated a relationship between the anterior pituitary and the nature of the estrous cycles in rats. Dresel (1935) discovered that PRL caused constant dioestrus in mice which was confirmed by Nathanson et al., 1937, and extended to rats (Lahr and Riddle, 1936). Nathanson and Fevold (1938) demonstrated that PRL could cause maintenance of corpus luteal function in normal mice. These ovarian responses were substantiated as prolactin activities by Evans et a1. (1941). KoviaEic (1962) developed a bioassay for PRL based upon the prolongation of dioestrus in mice. This method was later modified to utilize the corpora luteal cell nuclei count (CLCN) as a more reliable assay parameter (Nolthuis, 1963). A deciduoma assay was reported as yet another bioassay for PRL that same year (KoviaEic, 1963). However, it has recently been pr0posed, and accepted, to drop the "luteotropic" title used as a synonym for PRL (Short, 1972). Osmoregulation of serum sodium in fish (Ball and Ensor, 1967) and osmolar content of serum in humans (Buckman and Peake, 1973) has been established as a direct physiological role of PRL. Originally, Ensor and Ball (1968) proposed the name "Paralactin" for the PRL molecule in fish responsible for osmoregulatory actions on serum sodium balance of eury- haline teleosts in fresh water. Ovine prolactin was previously demon« strated to promote freshwater survival of hypophysectomized Poecilia latipinna, a cyprinodont fish (Ball and Olivereau, 1964). The effect of prolactin upon freshwater survival was first demonstrated by Pickford and Phillips (1959) and later confirmed as the single most important factor of the anterior and posterior pituitary principals in freshwater survival of yet another euryhaline teleost, Fundulus heteroclitus (the killifish, Pickford et al., 1965, 1966a,b). She also demonstrated that aldosterone, cortisol, extracts of corpuscles of stannius, hog renin, and parathyroid hormone did not inhibit the rapid fall in serum chlorine nor serum osmolality. Concurrently, Ball and Ensor (1965) demonstrated that ovine prolactin was effective in preventing the rapid decline of serum sodium'hihypophysectomized Poecilia latipinna placed in fresh water, which was later confirmed by those same workers (Ball and Ensor, 1967). Their later work demonstrated that serum calcium and potassium were not affected by either hypophysectomy, placement in fresh water or exogenous prolactin treatment. An assay for multispecies prolactin was then developed in hyp0physectomized Poecilia latipinna (a cyprinodont fish) in fresh water, measuring the maintenance of serum sodium levels as the response parameter (Ensor and Ball, 1968). Prolactin Biosynthesis The essential element in isolating a protein hormone is obtaining a highly potent and concentrated starting material at minimal expense. Therefore, the controls under which prolactin is produced jg_vivo might provide a key for obtaining potent starting material for isolation and purification studies. Neural regulation of PRL secretion was initially suggested by Selye (1934) finding that the suckling stimulus causes prolactin release from the adenohypophysis of the rat. Hypothalamic control of prolactin secretion was indicated by in_viv9_work of Desclin, 1950, 1956; Haun and Sawyer, 1960; Meites and Hopkins, 1960; Nicoll, Talwalker, and Meites, 1960; Everett, 1954, 1956; and Talwalker, Rather, and Meites, 1964. Ifl_vitro studies which support the jn_vivo work were done by Pasteels (1961a & b) and Meites, Kahn, and Nicoll (1961). These rat adenohypophyseal (AP) organ culture studies provided additional evidence for central nervous system inhibitory control of PRL secretion. PRL secretion occurred autonomously in AP's cultured in these systems. Upon the addition of hypothalamic extracts or comcultures of hypothalami and AP's, PRL secretion was inhibited (Pasteel, 1961b, 1962; Meites, Kahn, and Nicoll, 1961). Pasteels (1962) compared rat AP PRL release .1Q_vitro in the presence of hypothalamic and cerebral cortical extracts. PRL release was retarded in the presence of hypothalamic extracts as opposed to little or no inhibition of release under the influence of cerebral cortical extracts. Subsequent studies confirming these findings have been reviewed by Meites and Nicoll (1966), Bern and Nicoll (1968), Nicoll and Bern (1972), and Schally et a1. (1968, 1973). Steroids seem to markedly affect the jg,vixg_release of pituitary prolactin. Although gonadal steroids were first thought to inhibit hypophyseal secretions (Moore and Price, 1932), a different picture indeed has since been elucidated. Cytological changes in the anterior pituitary correlated with different phases of the estrus cycle were first demonstrated in the woodchuck by Rasmussen (1921). Later Charipper and Haterius (1930) confirmed these findings in the rat. Wolfe and Cleveland (1933) reported similar findings in the domestic dog and sow. It was suggested that different cell types and their numbers in the anterior pituitary were correlated with the phase of the estrus cycle (Cleveland and Wolfe, 1932). Severinghaus (1934) reported alterations in eosinophil counts in pituitaries of mature female rats treated with pregnancy urine. This report was soon after confirmed by Nelson (1934) who noted degranua lation of adenohypophyseal esoinophiles of male and female rats treated with oestrin. This evidence was demonstrated in the face of his earlier reports of increased chromophobic cell populations with a concomitant decrease in basophiles in pituitaries of animals under oestrin treatment (Nelson, 1933a & b). Nelson's work (1933a) further demonstrated that a 2-fold higher dose of oestrin or testis hormone was required to maintain normal AP cytology in castrate male rats than in females. Adenohypophy= seal eosinophile degranulation was demonstrated by Wolfe (1935) using large amounts of oestrin. The first reports demonstrating an increased pituitary PRL content in response to estrone or estradiol was by Reece and Turner (1936) for male rats and for female rats and male guinea pigs by these same authors (Reece and Turner, 1937). Testosterone proprionate and diethylstilbestrol were also shown to augment lactogenic hormone content ln_yixg_of female rats and male guinea pigs (Lewis and Turner. 1941). These findings were extended to the male rabbit that responded to estrone treatment ig_vivo (Meites and Turner, 1942). The‘jgjvivo stimulatory effects of estradiol, estrone, and testosterone on increased AP prolactin content were extended to yet another agent (norethynodrel, a synthetic progesterone) by Kahn and Baker (1964, 1966), and Kahn et al. (1965) in the female rat. More recently, Nicoll and Meites (1962) demon« strated a direct effect of estradiol upon female rat pituitary prolactin production in organ culture for periods of up to 3 weeks. These observ~ ations were confirmed by the demonstration that AP‘s from female rats pretreated with estradiol (50 pg estradiol benzoate/day for 6 days) (referred to as a "small dose") released more prolactin than noneprew treated female rat AP's in organ culture (Ratner et al., 1962). Gala and Reece (1964) were unable to confirm the direct effect of estradiol on AP prolactin release jn_vitro, but did reproduce the results of Ratner et al., 1963) using much lower doses of 1 to 10 ug/day for ten days. The increased synthesis and release of prolactin in vitro by AP's from estrogen-pretreated rats was further confirmed by using leucine-4,5-3H incorporation studies (Catt and Moffat, 1967; Macleod et al., 1969). Methods of Isolation Alterations in the pituitary protein profiles in relation to estraw diol and cortisol treatment were achieved through the use of analytical polyacrylamide disc gel electrophoresis (a—PAGE) (Lewis et al., 1965). This a-PAGE method offering higher resolving power than achieved before in analyzing mixtures of proteins was introduced by Ornstein and Davis (1962), Davis (1964), and Ornstein (1964). Soon after its introduction this technique was employed in isolating bovine and human growth hormone and displaying the protein hormone profiles of rat pituitary extracts (Lewis and Clark, 1963). The following year an apparatus for preparative polyacrylamide disc gel electrOphoresis (pwPAGE) was demonstrated by 10 Jovin et a1. (1964), which would revolutionize protein purification. Rat prolactin and growth hormone were the first hormones partially purie fied by the use of this technological advancement (Groves and Sells, 1968). These isolations, as all other prolactin purification procedures before this, extracted the hormone directly frompituitary glands (Kwa et al., 1967, Groves and Sells, 1968, Ellis et al., 1969, Neill and Reichert, 1971, for the rat; Cheever et al., 1969 for the mouse; Lewis et al., 1968 for bovine growth hormone and prolactin; Lewis et al., 1971 for human prolactin). Those utilizing organ culture medium as starting material fared better in their quantity and quality of hormone yields of rat PRL (Catt and Moffat, 1967, Gala, 1970, 1972; and human _ (growth hormone, Kohler et al., 1971). This technique appears to offer at least one of the major steps toward developing a method to economically isolate highly purified and biologically and immunologically potent rat prolactin in high yield. The questions remaining, then, are what are the optimal methods for obtaining good starting material, and how may it be concentrated, fractionated, and processed with minimum loss to yield a large amount of active hormone? MATERIALS AND METHODS Experimental Animals Sprague-Dawley adult male and female rats (Spartan Research Animals, Inc., Haslett, Mich.) were divided according to sex with four per cage and maintained on Purina Rat Chow and water ag_1ibitum. Caging environ- ment consisted of a room temperature of 23°C and a photo period of 14 hr. fluorescent light and 10 hours darkness, with the midpoint of each phase at noon and midnight, respectively. These animals were pretreated daily for five days with subcutaneous injections of 5 ug of estradiol benzoate (Nutritional Biochemical Company, Cleveland, Ohio) in corn oil. 0n the morning of day six, the rats were killed by decapitation and the pituia tary was immediately removed and placed in Medium 199 (Lot #528601, Difco Labs., Detroit, Mich.). Organ Culture Procedure The adenohypophysis (AP) was separated from the neurophypophysis, cut into 16 pieces (explants) approximately 1 mm3 in size, and cultured according to the methods of Fell and Robinson (1929), Chen (1954), as modified by Nicoll and Meites (1963), and Barnawell (1965). The explants were placed on polyester (Dacron) organdy rafts supported by stainless steel benches in 2.0 ml of Medium 199 containing 50 I.U./ml of penicillin (Penicillin G, Potassium, Lot #4440, Nutritional Biochemical Company, Cleveland, Ohio) and 5 ug/ml of insulin (Amorphous Beef Insulin, 11 12 Lot #PJ-657, Eli Lilly and Co., Indianapolis, Indiana). Cultures were incubated at 37°C in a humidified atmosphere of 95% 02, and 5% C0 , at a 2 pH of 7.4. At the end of every 48 hr. the medium was changed during culture periods of up to 6-8 days. The culture media were collected and pooled within sexes, and samples were taken for protein and prolactin analyses. Pooled medium and 48 hr. interval samples were quick frozen and stored at -20°C to await further treatment as shown in Figure 1. Concentration of Medium Optimal Conditions for Concentration: In order to determine the optimal conditions under which volume reduction of organ culture medium should be handled, a series of three conditions were tested. These con: ditions were determined from previous work on a similar apparatus employed in isolating human growth hormone (Lewis et al., 1969), and advice from Dr. Deal's lab (M.S.U.) on concentration of proteins similar to PRL in molecular weight (T. Massey, personal communication). A test batch of media, 150 ml in volume, was harvested from cultures of 110 AP's from animals varying in size, weight, pretreatment, and condition of health. The original volume of medium (150 ml) was mixed with a magnetic stirring device and Teflon stirring bar at 2°C to insure homogeneity of the mixture. This medium was aliquoted into three portions of 50 ml each. Each batch was separately sampled for protein and prolactin analyses. All of the concentration conditions tested utilized the Amicon Diaflo Apparatus (Model #52, Amicon Corporation, Lexington, Mass.) with a 65 m1 capacity and the Amicon UM=10 membrane (Lot #379, 43 mm diam.). This membrane possessed a molecular exclusion limit of approximately 10,000. 13 All volume reductions were carried out under N2 pressure at 2°C with rapid magnetic stirring. The three conditions tested were: (I) concenm tration at 55 PSI N2, (II) concentration at 20 PSI N , and (III) concenm 2 tration at 20 PSI followed by three washings. The latter washings cohsisbed of restoring the concentrated medium three times to its orig~ inal volume with cold deionized H20 and concentrating as before. In no instance was the magnetic stirring rapid enough to cause vortexing of the medium thereby risking protein denaturation. ‘ Actual Concentration Conditions: Media from five cultures, three from cultures of 50‘rat AP's each and two from cultures of 25 rat AP's each, were utilized as purification starting material. Each of these samples was separately quick~thawed in a 37°C water bath, with agitation, taking care to remove the tubes from the water bath while still contain= ing ice crystals. This procedure insured maintenance of media temperau ture at approximately 4°C. Once thawed, the media were separately cen= trifuged in polycarbonate tubes at 30,000 g's for 30 minutes (Sorval RC2-B, Head #SS-34) at 2°C. Both the supernatant and precipitate were sampled for prolactin content. The supernatant was then concentrated on an Amicon Diafiltration Apparatus (Model #52, Amicon Corporation, Lexington, Mass.) employing an Amicon UM=10 membrane (exclusion limit approx. 10,000, Lot #379, 43 mm dia., Amicon Corp.) at 20 PSI, N2 pres= sure with rapid magnetic stirring. These samples were concentrated by a factor of 3.5 leaving a concentrate never below 14.9 ml or above 22.0 ml in volume. Aliquots of the filtrate and concentrate were analyzed for protein and prolactin content while the remaining portions of each were quick frozen and held at ~20°C until used. Upon quick thawing, the 14 concentrates for preparative electrophoresis were made to contain 5% sucrose (wt./vol.) by the addition of dry, highly purified analytical grade reagent (Sucrose, Lot #50C-3080, Sigma Chemical Company, St. Louis, Mo.). Preparative Disc Gel Electrophoresis Preparative polyacrylamide disc gel electrophoresis (pwPAGE) was carried out according to the procedures of Jovin et a1. (1964). Buchler's "Poly-Prep-ZOO" column was employed with a Tris-HCl, Trismelycine discone tinuous buffer system at a running pH of 10.3. The equipment, reagents, and buffer systems used in these procedures are tabulated in Appendix B. All stock solutions were prepared in 20 gallon lots and stored at 2°C for periods no longer than sixty days. Analytical polyacrylamide disc gel electrophoresis (a-PAGE) as well as frequent pH checks were used to check the buffer solution consistency. Fresh preparative grade polyacrylamide was weighed out and prepared for gels of each run. The stacking and resolving gels consisted of a 62.5 ml volume of liquid each, and were polymerized at O-2°C. Following polymerization, the gels were of equal height at 35 mm each and 15.8 cm3 in area. The top of the stacking gel was washed with upper buffer solution, and then the apparatus was charged with buffers and set=up for the run. Organ culture medium concentrates now containing 5% sucrose were slowly applied to the top of the stacking gel through 1 mm 1.0. Tygon tubing with a peristaltic pump. Once applied, preparative electrophoresis was run at 40 ma constant current with a range of 200-400 volts at 0°C throughout. The elution buffer was circulated at a rate of 1.0 ml/minute while fractions were collected every five 15 minutes, i.e., 5 ml fractionation volumes. The elution buffer was monim tored throughout the run at 280 mu during fractionation and electron~ ically recorded. The fractions were again scanned on a Beckman DBG spectrOphotometer at 280 my and plotted. Aliquots of each fraction were taken for radioimmunoassay analysis of their prolactin content. Processing Column Eluants Dialysis: Fractions to be pooled for dialysis were determined on the basis of the protein elution patterns previously scanned and plotted. The dialysis tubing employed in this work had a 1.73 inch flat width and 1.125 inch inflated diameter (Lot #8a667E, Fisher Sci., Co.) and was boiled for 15 minutes in deionized, double distilled water, rinsed twice and held in 2°C deionized water for immediate use. Fractions were pooled directly into the dialysis bags and varied from 35 to 50 ml in volume depending on the column load. Fraction collector tubes were carefully aspirated free of solution using a pasteur pipette which minimized losses at this point. Each bag was placed in a 12 L., 2°C, circulating bath of deionized water for 48 hours. Aliquots were taken at this point for protein and prolactin analysis. Concentration of Dialyzed Fractions The pooled eluants were separately concentrated to a volume of 5~8 m1, irrespective of the starting volume, on an Amicon Diaflow Model 52 apparatus. The Amicon UMelO membrane was employed at 55 PSI N pressure 2 at 2°C with rapid magnetic stirring. Following volume reduction the filtrate and concentrate were sampled for later analysis. The concentrate 16 containing two deionized H20 washes, each of a 1.0 ml volume, of the dia- flow apparatus, was quick-frozen in 100 ml vessels and freezewdried in a glass column lyophilizer (Model K563000, Kontes Glass Co., Vineland, N.J.). Contents of the lyophilizing vessels were easily removed with a small sterile Spatula and placed in screw cap vials (cap loose) in vacuum desiccators containing Drierite and stored at room temperature. Deionized H20 washings of each vessel, each of 1.0 ml volume, were lyophilized and desiccated as before. Total dry yields were based upon weighings of the 72 hr desiccated materials, both initial and rinse lyophilization products, on a Cahn Electro Balance (Model G, Cahn InstUw ment Co., Paramount, California). Aliquots were taken for analysis as described in the following section. The remaining materials were stored under vacuum at -20°C. Analytical Technigues Bioassangrocedure: The intradermal pigeon cropesac assay for prolactin reported by Lyons (1937), as modified by Nicoll (1967), was used to determine the biological potency of isolated batches of rat prolactin. The paired assay procedure was employed as described by Nicoll and Meites (1963) using 6-week old white king pigeons (Cascade Pigeon Farm, Cascade, Mich.) weighing 370.7 :_8.9 gms (S.E. mean, n=lOO). Material from each isolated lot was carefully weighed out on a Cahn Electro Balance and dis~ solved in 0.9% NaCl solution at a pH of 8.0. Total doses of 1, 3, or 10 pg of lyophilized column fractions were injected intradermally on one side of the crop-sac while a known dose of ovine prolactin (NIHwP~SB, 28 I.U.lmg, National Institutes of Health, Bethesda, Maryland) was l7 injected over the opposite hemicrop. Four levels of ovine prolactin were employed as references in each assay: 1 pg, 3 pg, 10 ug and 20 pg. Assays were conducted over a three day period in which each pigeon was injected intradermally twice daily (each injection volume 0.1 ml), with control and experimental material on days one and two. On day three, approximately 20 hours following the last injection, the pigeons were killed by decapitation. The cropmsac of each pigeon was removed and the mucosal epithelium 4.0 cm in diameter around the center of the injece tion side on each hemicrOp was scraped free using a rounded elevator tipped probe (J. Sklar Mfg. Co., Long Island, N.Y.). The mucosal epia thelium from each hemicrop was placed in a preuweighed foil pan and dried overnight in an oven at 100°C. Following a drying period of 10-12 hours, each pan was placed in a desiccator, brought to room temperature, and weighed to the nearest 0.1 mg on a Mettler Balance (Model H18, Mettler Instrument Corp., Highstown, N.J.). Analysis of the hemicrop responses to the standards and unknowns are described under statistical methods. Radioimmunoassay Procedure The rat prolactin radioimmunoassay was performed as originally described by Niswender et a1. (1969) and modified as described in K. H. Lu et al. (1971). The reference preparations used in developing the dose response curves were NIAMD—RPwl at 1/7 ng/pl and KK~RP~1 at 1/7 ngfpl (see results). A comparison of the standards presently used for radiom immunoassay of rat prolactin was made on the basis of biological and immunological activity, and logit transformations (Rodbard et al., 1968; 18 Midgley et al., 1969) of standard inhibition curves as described in the results section. Lowry Protein Test Analysis of sample protein content was performed using the Lowry Protein Assay (Lowry et al., 1951). Bovine serum albumin (BSA), Fraction IV (Lot #A7044R, Grand Island Biological Co., Grand Island, N.Y.) was used as the reference preparation. Iodination of Rat Prolactin The radioiodination procedure has been previously described in f 125I Niswender et a1. (1969) and modified to incorporate 0.25 mCi 0 (New England Nuclear, Boston, Mass.). Iodinations were evaluated on the basis of plotting the activity eluted from 0.5 x 15 cm Bio-Gel P=60 (BioRad Laboratories, Rockville Centre, N.Y.) columns developed with 0.05 M phosphate buffered saline at a pH of 7.5. Eluants of one ml each were counted in a manual well type counter (Model #DSa202 (V), Nuclear~Chicago Corporation, Des Plaines, Illinois). Comparisons were made between our preparations and NIAMDoIml (courtesy of A. F. Parlow) and Ha10=10mB (courtesy of Dr. S. Ellis) rat prolactins. Analytical Polyacrylamide Disc Gel Electrgphoresis An estimate of purity of the isolated protein fractions was made on the basis of analytical polyacrylamide disc gel electrOphoresis (amPAGE). Preparations used in these analyses were KKwRP-I and H-lO=lO-B. The KK—RP-I material was one of the first isolated and purified lots of rat I9 prolactin from this work. Dr. Ellis's preparation, H-lO—lO-B, was used as the control in this procedure. The electrophoresis of these two 125I-labeled preparations was carried out on 0.5 x 6.5 cm, 7.5% gels, at 220 volts in a continuous buffer system of 1.538 M tris, 0.0713M Na EDTA, 2 and 0.2286 M boric acid. The pH of this system was 8.9 with a running temperature of 0°C. Each gel was layered with approximately 25 ul of the protein hormone iodination medium and transfer solution before the run commenced. Current was applied to the gels at a rate of l ma/gel for 15 minutes followed by 3 ma/gel for 67 minutes. The total run time was 82 minutes. Following electrophoresis, the gels were gently removed from the glass tubes, wrapped in Saran wrap, side by side, and X-ray film laid over the gels for a period of 3.0 minutes. The autoradiographs were then developed. Statistical Analyses Bioassay Results: The bioassay results were statistically treated by regression analysis employing the following equations: Y = B + 81 (Log X) Linear Regression 0 Equation and 0 = Log X Inverse Regression 1 Equation where: ..< N dried cropwsac tissue response (mg origin of the regression slope on Y axis W a II II 1 slope of the regression line >< ll dose of prolactin 20 The standard curves for each bioassay were plotted according to the rem sults of the regression analysis and an equation representing each curve was developed. Confidence intervals were computed for the origin (b0), slope (b1), mean levels of the standard (P-S~8, ovine prolactin), and the predictions from the regression equations (using the inverse regres= sion method for unknowns). Equations employed in developing these con= fidence intervals at the 95% level are given as follows: Confidence Interval on the Origin @ 95% level b0 = :_t a/2( n 2) (J 53 e/n 2 )y/r(1/n) + 322/55x Confidence Interval on the Slope @ 95% level b = :_(t a/Z, n~2) (Jsse/n-zm’ssx 1 Confidence Interval on Predictions from the Regression Curve at 95% level A 2 2 2 (ss+1.51(vO - Yo) ) g = (b1 - t a/Z, n+ m "3 e n + m + 3 _ 2. _.2 _ h - (Yo - v) /ssx — (n + m) (g/nm) m _. , ._ (sse +. Egvb Y')2 (h/g) c.1.=x+(b,oumm .cowpm_:omu mmo_ meucmscwm 8 mm _muop asp soc» Ho: .chFm ampm wasp op ump:n_cgpm mmmmop mm vmpmpznmg mLm mommog cowumwpp_sqo>_ use :owpmcpcmucoo\copumcupsmart mcpzoFFOL m:o_uumcm cmaszm m_mzpm_u mchoppom mcoppumcs cmaeam cowumcowpumcw\mpmmcogqocpuwpm m>wumcmamca mcwzoFFow cmswzm coppmcpcmucoo\covpmcp__mmwu newzoFFoe Ezwumz cowpmmsmvcucwu m:_20__o% Ezwumz Ezwvme chwmwco .H> .> .>H .HHH .HH .H “mommmuoca mcwzoppom ms“ ucmmmcamc mucmzcmm cw mamum asp .cowpm_0mw cwgomFoca mo mmmmum xwm pm :wuumFocq so >Lm>oomc A8 A: cam: .m mcsmwu 32 L___.|___L—L_—L\ o o o o o _c_>_ (D co N co AHBAOOBH lNBOUBd VI IV STEPS OF ISOLATION ‘11. ~ 33 96 PROTEIN RECOV ERY O. ON On 0* On .mpmgucmucou Ezwvws mezupau mo mmcuuma m scam >um>oumc :Pmuoca use :wuuwpocm .m mczmmm Ought... mwhst. w...wumsmqmga .n «snow; mum—232 mmsh mOkomifioo zo_._.oPm Lo; cams .o—_$oga :owuapm :wopoga m>wumgmamcm .m mesmwm . mum—232 .meH mOHOMJi—Oo 20:04?“ OON 00 On O¢ Om ON 0. O 20:045.“. 2.530%. _\. i No w 092 @ aonveuosev 37 analysis are shown in Figure 9 and tabulated in Appendix H. There appears to be identical agreement between the protein elution profile (measured at 280 mp absorption) and the radioimmunoassayable PRL analyses (Figure 9, Appendix H). This confirms the location of the PRL band and by taking only tubes 15 through 26 insures a more pure (uncontaminated by prealu bumins or albumins) product. It may be of interest to note that the yellow tag that PRL carries in pwPAGE appeared in all tubes registering a positive rat PRL RIA response. The color may be due to riboflavin. The yellow coloring was removed following 48 hr dialysis in deionized H20, but the PRL remained in the dialysis tubing. Isolation and Purification Efficiency: The efficiency of each of the steps (Figure l) of this procedure is reported in terms of mean percent loss (and total percent recovery) through each step (Figure 5, Appendix I). From the data collected one can see that the concentration step accounts for the greatest loss of PRL amounting to 14.5 :_l.3%. Losses in the other steps ranged from 3.2 to 7.4% (Appendix I). The cumulative mean percent recovery of five complete isolations is 67.6 :_3.3%. With the recent advent of Dupont's microfiber continuous flow dialysis and concentration systems it appears one could yet further improve on this efficiency. Pigeon Crop~Sac Bioassay The Standard Curves: The pigeon cropwsac bioassay “micro~method" (mnPCSA) was developed with four different levels of NIH~P~58 (ovine pro- lactin, 28.0 IU/mg, see Appendix AA). In all three of the bioassays in this study, no dose was evaluated on less than six hemicrops. Some were assayed over as many as nine test sites. Each assay was analyzed by .mmpwmoga :ovuzpm cwuomPoLn ecu :_muoga cszpoo m>wumgnqmgm .m mgamwu mum—232 mmDh cm on o a W - . .. a o m m . o .0 00. u ..... @ N 0 W + O M N O . . $ OON m m 2.530%. Sm . ( m m . 2.50m... . H m no mo“. .o.o . 7: com“ / w 39 linear regression analysis as previously described (Steele and Torrie, 1960). The crude data complete with body weights of each pigeon are tabulated in Appendices J, K, L, and M. The doseuresponse curves are graphically shown in Figure 10. The characteristics of each curve are summarized in Appendices J and K. The slopes of experiments I, II, and III are 13.9, 13.4, and 7.8, respectively (Appendix K). The bioassay correlation coefficients of experiments I, II and III are 0.85, 0.79, and 0.87. This indicates a close relationship between the cropmsac response and the log of the dose of prolactin (Appendix K). An index of precision used for comparing pigeon cr0p=sac bioassays in the past is lambda (A). Lambda represents the standard deviation of Y on X (analyzed by linear regression analysis) divided by the slope of the curve (Bliss, 1952). Each of these indices falls well under thbse reported by the National Institutes of Health (Appendix AA) and represents a very reli- able assay. Those A values are 0.256 (Exp. I), 0.311 (Exp. II), and 0.287 for experiment III. Each of the pigeon cropasac responses were analyzed by covariate analysis with body weight and body weight to the threemquarter power (Finney, 1964). The results of these analyses did not aid in linearizing the pigeon responses and therefore are not reported. Weighted regression analyses were also run on each of the bioassay dose= response curves aften Steele and Torrie (1960). The heterogeneous vari» ance (tested by Hartley's Fumax test (1950) encountered in experiment one and two were too great to be dealt with by this method (J. L. Gill, per= sonal communication). Therefore, a method of eliminating the heteror geneous variance which existed in experiments one and two, but not three, .was sought. If the 20 pg dose level responses were dropped from the 40 .mpcmswgmaxm mags“ Low mm>g=u ugoucopm Ammmmown cam-no.0 :oomwm .o_ mgamw. z....0<...0mn. “.0 wmoo 0.0N 0.0. 0.m 0.. — — — — VK‘K + O :x oo...m...+¢.o_.: 1. . “Ia 5-8-... szmgmmamm o 0 0m 3 2x 8.51m. . 37% 414 0 5.6.8. fiezmzauaxm . H 2x oo...m.n..m.n..% I i 00. m ENE. H 3535 m a i on. w 3 H 1 o.o~ w. 3 H i 0.3 m mm>m30 1M. omoEmc mcwzo__o. gmapwm zepmn m_QEmm mpmcupwm c. 6.685 mo manpo> cmppww m>onm mpasom mpmgpcmocou c. 6.685 yo mE:_o> oom pm nFm; LmnEmso spvz N:w\mn_ cw mcammmga :mmoguwz ugmucmpm a ma w=cm Fuamuoz “may meuwcw comm AAAAAM (U .Q U 'U Q) Q— m VVVVVW m.~ m.o m.o o o N.Nm 8* .LHFP. m.o F m.F o o 0.0m m» .cpp_. m.o N N.N o o 0.0m Na .Lgpw. m.N m 8.3 o o m.om p4 .Lp_w. _m— m.~o_ we m.mn Kw N.NP m.m~ Am..ocou m.NF a ¢.m o _.o m.mm .cpPF. mm. N.Pm_ we we mm N.P_ m.mF .ocou N.mp m _.m o ~.o m.mm A.V.Lppw. may m.~mp Na m.om No e.m o._m Am..ucou w.~m oop m.Fm_ oo_ o.¢~ om :owpmcucmocou me\mn a me x as APE. :mucm : o; . o; cpm o; :P on c; we: o m use a a .p m\ P a flow,. u a Anv.u _ a on. p > P m ucmpcoo Ezwumz monHHozou hzmmmmmHo mmoz: zzHomz mm:b.:u z->J>4> h-h-h-b wwww O O 0103 \Isoxo—o wNN—J Noww —JNU‘IU‘I db ODOU'IV wbmd NOWO-b 1"de KK-RP~4 4.52 :_0.07 1.7521 ~2.5912 .99 NIAMD-RP~1 3.68 :_0.14 1.7145 -2.9854 .99 O NIAMDuI-1 2.64 :_0.02 1.2066 ~2.8702 .99 H~10~10-B 5.18 i 0.07 1.8802 w2.6322 .99 01010101 NNNN 00000000 0 O-dw-fl 03050503 NOVV oooono hNON oomuoo (l) From four assays in quadruplicate. 77 APPENDIX 5 SUMMARY TABLE OF THE ANALYSES OF BIOLOGICAL AND IMMUNOLOGICAL ACTIVITY OF ISOLATED COLUMN FRACTIONS Biological (1) Immunological (2) Preparation Potency (IU/mg) Potency (ng) KK-RP-I 29.5 3.35 1 0.11 KK-RP-II 27.1 4.23 i. 0.06 KK-RP-Z 25.2 4.24 _+_ 0.05 KK-RP-3 27.0 3.95 i 0.08 KK-RP-4 26.9 4.25 i 0.07 (l) The reference preparation used consistently throughout all assays at identical total doses was NIH-P-S8, ovine prolactin with a potency of 28.0 I.U./mg. These responses are crude means tranSformed by inverse linear regression analysis. (2) Fifty percent binding point developed from the means of four assays computed by regression analysis. NIAMD-RP-l was used as the reference comparison in all cases. 78 APPENDIX T SUMMARY TABLE OF THE ANALYSES OF BIOLOGICAL AND IMMUNOLOGICAL ACTIVITY OF ISOLATED COLUMN FRACTIONS Biological (1) Immunological (2) Preparation Potency (IU/mg) Potency (ng) KK-RP-I 27.7 3.35 i 0.11 KK-RP-II 27.7 4.23 :_0.06 KK-RP-Z 30.5 4.24 i 0.05 KK-RP-3 27.1 3.95 2‘. 0.08 KK~RP~4 32.2 4.52 :_0.07 (l) The reference preparation used consistently throughout all assays at identical total doses was NIH-P-S8, ovine prolactin with a potency of 28.0 I.U./mg. These are means of the inverse regression analysis transformed crop-sac responses. (2) Fifty percent binding point developed from the means of four assays computed by regression analysis. NIAMDwRP-l was used as the reference comparison in all cases. 79 APPENDIX U COMPARISON OF BIOLOGICAL AND IMMUNOLOGICAL ACTIVITIES 0F COLUMN FRACTIONS WITH AVAILABLE RAT PROLACTINS » Biological (1) Immunological _Pneparation Potency (I.U./mg) Potency (ng) KK—RP-I 29.5 3.35 I. 0.11 KK-RP-II 27.l 4.23 :_0.06 KK—RP-Z 25.2 4.24 :_0.05 KKaRP-3 27.0 3.95 :_0.08 KK-RP-4 26.4 4.52 i 0.07 NIAMD-RP—l 29.5 3.68 :_O.l4 NIAMD-I-l 38.4 2.64 :_0.02 H-lO-lO-B 25.5 5.18 :_0.07 (1) Crude means transformed by inverse linear regression analysis after Ostle (l963). 80 APPENDIX V COMPARISON OF BIOLOGICAL AND IMMUNOLOGICAL ACTIVITIES 0F COLUMN FRACTIONS WITH AVAILABLE RAT PROLACTINS 1(1) Biologica Immunological Preparation Potency (I.U./mg) Potency (ng) KK-RP-I 27.7 3.35 2: 0.11 KKwRP-II 27.7 4.23 :_0.06 KK-RP-z 30.5 4.24 i 0.05 KK-RP-3 27.1 3.95 _t 0.08 KK—RP-4 32.2 4.52 _+_ 0.07 NIAMD-RP-l 24.2 3.68 i 0.14 NIAMD-I-l 28.5 2.64 .t 0.02 H-lO-lO-B 28.6 5.18 3; 0.07 (1) Means of inverse linear regression analysis transformed cropwsac reSponses. 81 APPENDIX N RELATIVE COMPARISONS OF THE ISOLATED COLUMN FRACTIONS AND RAT PROLACTIN STANDARDS Biol. (1) Immunol. (2) Biol. Pot. (3) Preparation Index Index Immunol. Pot. KK-RP-I 1.00 0.91 1.10 KK-RP-II 0.92 1.15 0.80 KK-RP-2 0.85 1.15 0.74 KK-RP-3 0.92 1.07 0.86 KK-RP-4 0.90 1.23 0.73 NIAMD-RP-l 1.00 1.00 1.00 NIAMD-I-l 1.30 0.72 1.81 H-10-10-B 0.86 1.41 0.61 (1) This index was developed by dividing all biological potencies by that of the national standard for rat prolactin. NIAMDuRPul. The crude means, transformed by inverse linear regression analysis, were used to make these computations. (2) This index was computed by dividing all RIA fifty percent binding means by that of the national standard for rat prolactin, NIAMDaRPal. (3) An index computed by dividing the biological index by the immunow logical index. 82 APPENDIX X RELATIVE COMPARISONS OF THE ISOLATED COLUMN FRACTIONS AND RAT PROLACTIN STANDARDS Biol. (1) Immunol.(2) Biol. Pot. (3) Preparation Index Index Immunol. Pot. KK-RP-I 1.15 0.91 1.26 KK-RP-II 1.15 1.15 1.00 KK-RP-2 1.26 1.15 1.10 KK-RP-3 1.12 1.07 1.05 KK-RP-4 1.33 1.23 1.08 NIAMD-RPul 1.00 1.00 1.00 NIAMD-I-l 1.18 0.72 1.64 H=10-10~8 1.18 1.41 N 0.84 (1) This index was developed by dividing all biological potencies by that of the national standard for rat prolactin, NIAMDnRPal. The means of the inverse linear regression transformed crop-sac responses were used to make these computations. (2) This index was computed by dividing all RIA fifty percent binding means by that of the national standard for rat prolactin, NIAMDaRPml. (3) An index computed by dividing the biological index by the immunological index. 83 APPENDIX Y SUMMARY OF BIOLOGICAL AND IMMUNOLOGICAL ACTIVITIES 0F COLUMN FRACTIONS WITH AVAILABLE RAT PROLACTINS Preparation Biological Immunological Potency (I.U./mg) Potency (ng) 31‘ 2 c.1.( KK-RP-I 27.7 12.8 - 49.6 3.35 :_0.11 KK-RP-II 27.7 11.5 - 53.0 4.23 :_0.06 KK-RP-Z 25.2 11.6 - 53.6 4.24 :_0.05 KK-RP-3 26.9 14.0 - 90.6 3.95 i 0.08 KK-RP-4 26.4 13.8 «104.3 4.52 :_0.07 NIAMD-RPc1 24.2 8.7 - 45.6 3.68 :_O.14 NIAMD-I-1 28.5 10.8 a 56.6 2.64 :_0.02 H-10-10-B 25.5 12.3 - 41.4 5.18 i 0.07 (1) C.I. @ 95% level by linear regression analysis, inverse method. (2) Means and 95% confidence intervals (C.I.'s) from Appendix 0. 4 These means were chosen as being the highest estimate of biological potency but with the most reliable 95% C.I.'s. 84 APPENDIX Z SUMMARY OF RELATIVE COMPARISONS OF THE ISOLATED COLUMN FRACTIONS AND RAT PROLACTIN STANDARDS 8101. (1) Immunol. (2) 3101. Pot.(3) Preparation Index Index Immunol. Index KK-RP—I 1.14 0.91 1.25 KK-RP-II 1.14 1.15 0.99 KK-RP-Z 1.04 1.15 0.90 KK-RP-3 1.11 1.07 1.04 KK—RP-4 1.09 1.23 0.89 NIAMD-RP-l 1.00 1.00 1.00 NIAMD-I-l 1.18 0.72 1.64 H-10-10-B 1.05 1.41 0.74 (1) This index was developed by dividing all biological potencies by that of the national standard for rat prolactin. NIAMD-RP-l. (2) This index was computed by dividing all RIA fifty percent binding means by that of the national standard for rat prolactin, NIAMD=RP~1. (3) An index computed by dividing the biological index by the immunow logical index. REFERENCES Arai, Y. and T. H. Lee. A double-antibody ratioimmunoassay procedure for ovine prolactin. Endocr. 81(5):1041-1046, 1967. Asdell, S. A. Recent development in the field of sex hormones. Cornell Vet. 20:147-152. 1931. Ball, J. N. and D. M. Ensor. Effect of prolactin on plasma sodium in the teleost, Poecilia latipinna. J. Endocr. 32(2):269~270, 1965. Ball, J. N. and D. M. Ensor. 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