The Nature of a Nematode Growth Factor. Iii Growth and Maturation of Caenorhabditis Briggsae on Protein- Haemin Co-Precipitates By
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THE NATURE OF A NEMATODE GROWTH FACTOR. III GROWTH AND MATURATION OF CAENORHABDITIS BRIGGSAE ON PROTEIN- HAEMIN CO-PRECIPITATES BY J. R. VANFLETEREN Laboratoria voor Morfologie en Systematiek,Museum voor Dierkunde, Rijksuniversiteit Gent, Ledeganckstraat35, B-9000 Gent (Belgium) The growth promoting activity of protein-haemin co-precipitates from ferritin, apoferritin, transferrin, bovine serum albumin, conalbumin and egg white on maturation and reproduction of C. briggsae has been evaluated. Ferritin, apoferritin and transferrin were found to be biologically highly active in the presence of haemin. Bovine serum albumin, conalbumin and egg white were slightly active. Maturation and reproduction of C. briggsae on the coagulates from bovine serum albumin and egg white were nearly independent of the dose administered, probably because the limited availability of haemin from these coagulates permits but slow growth, even in the presence of abundant proteinaceous material. Bovine serum albumin, egg white and conalbumin failed to support continuous growth of C. briggsae. It is supposed that the limited availability of haemin from these coagulates inhibits normal maturation and reproduction of the F1 progeny. These experiments clearly demonstrate the requirement for particulate haem. The requirement for specific protein is not supported. Proteins, especially globulins, show a marked tendency to adsorb metal ions on their surface. Metallo-proteins such as ferritin, transferrin and conalbumin speci- fically bind Fe3+ ions and perhaps might absorb haemin through interaction with the central Fe3+ ion of the latter. Interaction might also be established by Van der Waals forces between the porphyrin nucleus and apolar groups of the protein. Globin and serum albumin have been reported to form complexes with porphy- rins through the carboxylic acid side chains of the latter (Falk, 1964). Buecher et al. (1970), in a study of the growth promoting activity of several commercially available proteins in defined medium containing haemin, reported the following supplements to be active: «, f3 and y-globulin, {3-lipoprotein, haemo- globin, f3-lactoglobulin, albumin (bovine and egg), insulin and soy peptone. Yet, in general, the dose at which this material was tested was very high (10-20 mg protein/ml) and the response very low (maturation times ranging from 9 to 20 days and a final population from 20 to 100 nematodes) except for y-globulin (best maturation time being 5 days, best final population some 1 000 worms). Insulin and TMV protein, both also slightly active in the presence of sterols and haemin, have been investigated in some more detail because these proteins are chemically fully defined (Buecher et al., 1971). I believe that the poor growth obtained with these proteins is mainly due to 104 the choice of inappropriate material and to the lack of optimal activation. In the present study, experiments are described in which iron binding proteins were dissolved in the presence of haemin chloride and subsequently carefully pre- cipitated and assayed in defined medium containing haemin and sterols. Fast growth and maturation of C. brigg.rae on such co-precipitates suggests that this organism has no nutritional requirement for a specific protein. MATERIALSAND METHODS The growth conditions and biological assay were as described previously (Van- fleteren, 1975a, 1975b). Preparation of protein-haemin co-precipilates Ferritin-haemin co-precipitate. Ferritin (Serva, 2 X cryst., Cd free, electron- microscopically pure, 10% sterile solution) was diluted in defined medium (pH 5.9, containing sterols at 50 flg/ml and haemin at 10 pg/ml) to a stock concen- tration of 10 mg ferritin/ml. Strong H:,,P04 was added aseptically until a heavy precipitation occurred (pH nearly 3.5) and was subsequently neutralized with KOH (final pH 6.4). Serial dilutions were made afterwards in defined medium (sterols at 50 fig/ml and haemin at 10 flg/ml), pH 5.9. Suitable precipitates were also formed by acetone precipitation. Ferritin solution was diluted with sterile distilled water and haemin chloride to obtain the final concentration: 20 mg ferritin/ml and 200 fig haemin/ml. The pH was adjusted at 4.4 and cold acetone was added until the protein precipitated completely. The precipitate was collected by centrifugation and washed two times with sterile distilled water. Some loss of material is inevitable because a fraction of the precipi- tate dissolves. The remaining precipitate was then resuspended in sterile distilled water and dispersed in a Bransonic ultrasonic cleaner until a fine and homogeneous suspension was formed. Protein was determined by weighing. Finally, the suspen- sion was diluted to the desired concentrations in defined medium (pH 5.9) con- taining sterols at 50 ug/ml and haemin at 10 ILg/ml. Apoferritin-haemin co-ps°ecipitate. 90 ing apoferritin from horse spleen (Cal- biochem, A grade) was dissolved in 4.5 ml haemin chloride from stock solution (1 mg/ml) and sterilized by Millipore filtration (0.30 Sterile 1 M HCI was 'added to 0.5 ml filtrate to induce precipitation. The precipitate was further diluted with 4.5 ml defined medium (containing sterols at 50 fig/ml) , pH 4.45 and 5 ml defined medium (sterols at 50flg/ml), pH 6.51 in this order. The final con- centrations then were: apoferritin 1 mg/ml and haemin 50 tkglml and the final pH was 5.92. Serial dilutions were made in defined medium (sterols at 50 /tg/ml and haemin at 10flg/ml) pH 5.4. Tr?zn.r f eYriyl-haemin-?ntit??an.r f erri?aprecipitate. Transferrin could not be precipi- tated in the proper conditions for bioassay and was therefore assayed in the .