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Dense Crithidia Growth and Heme Sparing: Relation to Fe, Cu, Mo Chelation *
Author(s) -
SHAPIRO ANNA,
HUTNER S. H.,
KATZ LILLIAN,
BACCHI C. J.,
O'CONNELL TAMBURRO KATHLEEN,
BAKER HERMAN
Publication year - 1978
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1978.tb04180.x
Subject(s) - crithidia fasciculata , heme , biochemistry , chemistry , sulfosalicylic acid , histidine , growth medium , amino acid , chemically defined medium , myoglobin , biology , chromatography , in vitro , dna , bacteria , genetics , enzyme
SYNOPSIS. Heme, intrinsically required by Trypanosomatidae, is unstable, especially in conventional alkaline (pH 7.2–8.0) media. Low solubility of heme in a pH 6.5 basal medium (developed to assay biopterin with Crithidia fasciculata ) posed a problem: in media acidified during growth because of glycolysis, heme precipitated, perhaps contributed to acid‐limited growth and interfered with densitometric estimation of growth. The remedy was to: replace glucose with less rapidly metabolized mannitol; distribute media in thin layers to promote oxidation of acetate, fumarate, and malate (presumably leaving an alkaline residue); and buffer heavily with histidine + Good zwitterionic buffers, and superimpcse physiological buffering by arginine + asparagine whose catabolism appeared to yield an excess of NH + 4 over acid. Thereupon, Fe and Cu deficiencies sharply limited growth in the medium whose main chelators were: ( a ) 2,3–dihydroxybenzoic + 5‐sulfosalicylic acids (which preferentially bind transitional elements at their higher valences; ( b ) malic and gluconic acids; and ( c ) histidine. With unconventionally heightened concentrations of Fe, Cu, and Mo (the latter serving as Cu buffer as well as nutrient per se), the hemin concentration could be lowered, widening the margin of safety for heme solubility. Growth then reached 1.4 × 10 8 cell/ml. This medium may serve to screen for ligands promoting uptake or release of Fe and Cu. The increased growth is a step towards improving the assay medium for biopterin and practical use of Crithidia to assay several B vitamins and essential amino acids for metazoa.