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A Distinctive Subcellular Particle of Mammalian Melanocytes

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A Distinctive Subcellular Particle of Mammalian Melanocytes View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector THE MELANOSOME: A DISTINCTIVE SUBCELLTJLAR PARTICLE OF MAMMALIAN MELANOCYTES AND THE SITE OF MELANOGENESIS* M. SEIJI, M.D., Pxs.D.,t T. B. FITZPATRICK, M.D., Pss.D.t AND M. S. C. BIRBECK, M.A.t Tyrosinase from different sources has differentthat the melanized cytoplasmic particles pres- characteristics (1); that obtained from plantent in suspensions of mouse melanoma are tissue can usually be prepared in colloidal solu-modified mitochondria (6,7) because they con- tion, while that obtained from mammalian tissuetain, in addition to melanin, not only cytochrome is held by ultramicroscopic, cytoplasmic particles.oxidase, succinic oxidase in markedly varying Localization of tyrosinase activity in suspensionsdegrees and eytochrome C, but also tyrosinase. of cell particles was first reported by HerrmannThe need for resolution of these differences of and Boss (2) in preparations of the ciliary bodyopinion has been emphasized by Dalton (8) and of the bovine eye. Later, Lerner et al. (3) alsoBirbeck (9) who make a plea for correlation of reported that in their differential-centrifugationthe findings in electronmicrographs with chemical studies of suspensions of homogenized Harding-data obtained from melanin granules and mito- Passey mouse melanoma, tyrosinase activity wasehondria after centrifugation. Such a study has found to be present in the particulate fraction.been carried out with experimental procedures They conjectured that the structural elementsimproved so as to assure the most effective in this fraction were "microsomes, or particlespossible separation of melanin granules and the size of microsomes, which were formerlymitochondria. A preliminary report of these part of larger aggregates that were dispersedresults has already appeared (10) and the follow- during the experiments." ing experiments provide conclusive data in There seem to be two contrasting beliefssupport of the concept that melanin granules about the relationship between mitochondria andand mitoehondria are disparate, subeellular melanin granules in melanin-forming cells: oneparticles in the mammalian melanocyte. is based on morphological findings in electron- micrographs, the other on histochemical and MATERIALS AND METHODS biochemical observations. Studies by electron- 1. Preparation of the Specific Gravity-Gradient microscopy have led to the belief that melanin Tubes granules are formed from cytoplasmic organelles, Tubes of the Spinco swinging-bucket rotor, SW such as the Golgi apparatus or "pigment-forming39-L, were prepared by layering 0.5 ml. of eight centers" (4, 5), and the granule is melanizeddifferent concentrations of sucrose solution in gradually. The proponents of this view concludeserial order, with the most concentrated layer at that melanin granules and mitochondria arethe bottom of the tube. They were then allowed to disparate cytoplasmic particles, since no evidencestand overnight (between 8 and 20 hours) so that has been published to show that there existthe gradient might become smooth; at the end of structures intermediate between mitoehondriathis interval, 1 ml. of freshly prepared "large- granule suspension" was layered carefully over the and melanin granules and since the membranetop of each tube. For low-density studies, the structure of pre-melanin granules differs fromsucrose concentrations used were 2.0 M, 1.8 M, that of mitochondria. Per contra, the interpreta-1.6M, 1.55M, 1.5M, 1.4M, 1.2M, 1.OM (Fig. 1 a) tion of biochemical assays and vital staining isfor high-density studies, they were 2.6 M, 2.4 M, *Fromthe tDepartment of Dermatology of2.2 M, 2.0 M, 1.6 M, 1.55M, and 1.5 M (Fig. 2 a) the Harvard Medical School at the Massachusetts General Hospital, Boston 14, Massachusetts and 2. Preparation of "Large-Granule Suspension" the tChester Beatty Research Institute, In- From Mouse Melanoma stitute of Cancer Research, Royal Cancer Hos- B-lb mouse melanoma was serially transplanted pital, London, England. Presented at the Twenty-first Annual Meetingin C-57 strain mice. The entire, actively growing of the Society for Investigative Dermatology,melanoma was excised wben it reached 1—1.5 cm. Inc., Miami Beach, Florida, June 13, 1960. in diameter, and promptly homogenized in 0.3 M This investigation was supported in part by the Damon Runyon Cancer Fund and U. S. Publicsucrose at about 0°C. All subsequent processing Health Service Research Grant CY-5010. took place in a cold environment (about 3° C.). 243 244 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY PREPARATION OF "LARGE-GRANULE SUSPENSION" Fraction Large number 10 mlgranu• susPens'on. B-16 MOUSE MELANOMA in 10 M 05ml. 2 S vole. isotonic sucrose 12 M 05ml. homoginized a'14 M 05 ml. :3 15 N 05ml.Sucrose SI 0 solution. centrifuged ISSM05 ml. 4 710 ag for 10 mm. 16N 05 ml. iBM OS ml. S Sediment SUPERNATANT cells, nuclei, I 2OM OS ml. 6 celldebrisl 11,000 ag for 10 mm. l.a Lb SEDIMENT Supernatant Fia. 1. a. Density-gradient tube before cen- I (microsomeal trifugation, showing the layering of eight different resuspended concentrations of sucrose solution and the large- 15, 000 xg for 10 mm. granule suspension (in 0.3 M sucrose). The gradi- ent was prepared 18 hours in advance; the SEDIMENT Supe rnatant large-granule suspension was added immediately before centrifugation. resuspended in b. Fractions 1 through 6 as they appear in the isotonic sucrose gradient tube after ultracentrifugation at 103,000 X g for 1 hour in a horizontal rotor. The four "LARGE-GRANULE SUSPENSION" opaque fractions and the places where the tube (mitochondria, melanin granules) was cut are shown diagrammatically. FIG. 3. Preparation of large-granule suspen. shIn. FRACTION NUMBER 3.Separation of the Various Fractions After LARGE l.OmI GRANULE Centrifuqation SUSPENSION 0.Sml H At the end of ccntrifugation, after the position and thickness of the various strata in each tube _0.5 ml 2 .6 0.5 ml had been recorded (Fig. 1, 2), the strata (fractions) 0.5 ml were separated by means of a specially designed LB 1 2.0 0.5 ml centrifuge-tube cutter (10). Figs. 1 b and 2 b show 2.2 0.5ml where the tube was cut to permit collection of the 2.4 0.5 ml individual fractions and how the fractions were numbered. 2.6 0.5 ml 5 a. 4. Enzyme Assays FIG. 2. a. Tube containing modified density- Determinations of succinoxidase and gluta- gradient with large-granule suspension before centrifugation; the gradient consists of eightmatc-oxidasc content were made immediately different concentrations of sucrose solution, andafter separation of the fractions; determinations was prepared 18 hours previous to centrifugation.of tyrosinase and protein-nitrogen content were The large-granule suspension in 0.3 M sucrosealways completed within 48 hours after separation. was added immediately before centrifugation. b. Fractions 1 through 5 as they appear in the Succinoxidase content of the fractions was gradient tube after ultracentrifugation at 103,000estimated respirometrically by measuring oxygen X g for 1 hour. The two opaque regions and theconsumption in the presence of cytochromc C, as places where the tube was cut are shown diagram-described by Umbreit (11); some estimations were matically. also made with ATP (2.8)< 10 M) and DPN (1.5 X 1)1' M) as co-factors. The homogenate was centrifuged at 700 X g for 10 Glutamate oxidase was estimated respiromctri- minutes. The resulting, low-speed supernatant,cally by measuring oxygen uptake in the reaction when centrifuged at 11,000 X g for 10 minutes,system: potassium phosphate buffer, pH 7.5 (1.2 yielded a sediment which was resuspended in 0.3X 10 M); ATP (2.5 X 102 M); DPN (1.5 X 1O M sucrose and reccntrifugcd at 15,000 X g for 10M); nicotinamidc (1.2 )< 10_2 M); MgC12 (8 X minutes. This sediment was again suspended in 0.3 103); cytochrome C (8.3 X 10—i); and glutamic M sucrose to make the "large-granule" prepa-acid (1 X 102 M) in the presence of KOH in the ration which was used as starting material forcenter well. specific gravity-gradient ccntrifugation (Fig. 3). Tyrosinase content was estimated respiromct- THE MELANOSOME AND THE SITE OF MELANOGENESIS 245 rically by measuring oxygen consumption, using 00 - a 10:1 mixture of L-tyrosine and L-dopa as sub- • S Protein—Nitrogen x strate (1.77 micromols) in the M/10 phosphate 90 - X X Tyrosinase buffer (pH 6.8) (12). v——.-v Succinoxidase 80 - 5. Protein-Nitrogen Determination 70 - V I Protein was precipitated by adding 10 per cent I' I trichioroacetic acid to each sample. The precipi- I' I tate was spun down, resuspended in tricbloroace- 60 - , tic acid and centrifuged once more. The nitrogen- I' I content of this sediment was determined after di- Ca50 - \ gestion in a micro-Kjeldahl flask and steam dis- Lj tillation. 40 - 6. Preparotion of Retinal-Pigment Epithelium of 30 - the Chick Embryo 20 - Retinal pigment-epithelium, dissected from the eyes of Rhode-Island-Red chick embryos, was homogenized in 0.3 M sucrose. The homogenate 10 ci A was centrifuged at 300 X g for 5 minutes and suc- I_.— cessive centrifugations were carried out twice more with the respective supernatants. The last 2 3 4 5 supernatant was recentrifuged at 300 X g for 5 PR4CIO/V minutes and centrifuged once more at 11,000 X g Fin.4. Recovery of protein-nitrogen, tyrosin- ase, and succinic oxidase in Fractions 1 through 6 for 10 minutes. The resulting sediment was thenof large-granule suspension of B-16 mouse mela- resuspended in 0.3 M sucrose and recentrifuged atnoma following density-gradient centrifugation. 11,000 1< g for 10 minutes. Finally, the sedimentPercentages are calculated in terms of the sum of was suspended once more in 0.3 M sucrose to formthe amounts recovered in all fractions.
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