Radiation inactivation analysis of amino acid transport systems in Neurospora crass

DeBusk, A. G.

DeBusk, B. G.

Mallon, J.

Fungal Genetics Reports

English

B. G. DeBusk

J. Mallon

A. G. DeBusk

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Radiation inactivation analysis of amino acid transport systems in Neurospora crassa

Kansas State University

Fungal Genetics Reports Volume 8 Article 3 Radiation inactivation analysis of amino acid transport systems in Neurospora crassa B. G. DeBusk J. Mallon A. G. DeBusk Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation DeBusk, B. G., J. Mallon, and A.G. DeBusk (1965) "Radiation inactivation analysis of amino acid transport systems in Neurospora crassa," Fungal Genetics Reports: Vol. 8, Article 3. https://doi.org/10.4148/1941-4765.2076 This Research Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Radiation inactivation analysis of amino acid transport systems in Neurospora crassa Abstract Radiation inactivation analysis of amino acid transport systems in Neurospora crassa This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol8/iss1/3 RESEARCH NOTESCarsiotis, M., A. M. Lacy and D. B.  Fankhauser. As reported earlier (Carsiotis and Lacy 1965 J. Bacterial.  89:Increased activity  of the first two enzymes of 1472),  the last two  enzymes of the tryptophon  biosynthetictryptophan biosynthesis. Pathway  ( indoleglyceml  phosphate synthetose  and  tryptophansynthetase ) ore elevated +wo-fold or rmre  in all histidinemutants of Neurosp~ro  crassa.We have  now  comPTZZsurveying  these same  mutants and found a similar increase in the first two enzymes of the Pathway(~nthronilate  synthetase  and PR-transferare), thereby providing proof that the entire pathway is elevated in these mutows.The mechanism of this elevation is currently under investigation. - - - Department of Microbiology, University ofCincinnati College of Medicine, Cincinnati, Ohio and  Department of Biological Sciences, Goucher College, Towson,  MarylandCarsiotis, M. and A. Meyers. Decreased activity of A reinvestigation of the effect of zinc deficiency rated byall tryptaphan biosynthetic enzymes due to zinc deficiency. Nasm  et al. ( 1951  J. Biol.  Chem. l88:397)  has  revealed- -that all enzymes of the tryptophan biosynthetic pathway  aredecreased two- to five-fold when zinc is omitted from the growth medium (Vogel’s Medium N). The effect is mare  markedin a histidine mutant than  in the wild type. An explanation of this effect is being -ought currently. - - - Department ofMicrobiology, University of Cincinnati College of Medicine, Cincinnati, Ohio.DeBusk,  B. G., J. Mollon and  A. G. DeQusk. Permew  systems, i.e., enzymes res+wnsible  for the transferRadiation inactivation analysis of amino acid transport of met&liter into?K  cell, con  be studied only in an  intactsystems in Neumspora  crasso. cell. Therefore, standard methods of molecular weight deter-mination are  ruled wt. However, since we were interested inobtaining approximate molecular weights for some amino  acid permease  systems, we have used radiation inactivation of theenzymes as a means to this end.Neurospara  crassa  conidia were exposed to X-rays produced by a 3MEV Van der Graaf  accelerator operating at  5Qamperes  for varyingtimes,  giving tot.1 dosages from 300,000 to 2,500,OOO  rads.  After exposure to X-radiation, the cellswere examined for the effect of irradiation  on the transport  of amino  acids. Four amino acids were employed; phenylalanine,leucine, tryptophan and  glycine, with particular emphasis on phenylalanine. In each case, the irradiated cells were com-pared to control cells which had been handled identically except that they were not irradiated.The dota obtained from thirteen experiments employing phenylalaninewere averaged and plotted in the accompanying figure. The fraction of the ,,remoining  activity, as compared to the control, is plotted as the ordinate;the dosage as the abscissa. If the eqwtion  in A/A0 = - (cons+on+)(dosoge), 2 ;where A/A0 =remaining  activity holds, a straight line should be obtained. PhWl~lOlO”i”~It can be seen from the figure that such is not the case for phenylalanine, Inor  was it the case  for the other three amino  acids tested.This non-linearity would indicate that more thcln one enzyme is involved ,5in the transport of the amino acids  or that mare  than one “hit” is necessaryto inactivate  the transport system. If the straight line portion of the curveis extrapolated back  to the zero dosage, the intercept values for phenyl-alanine,  tryptophon and  leucine are near  two. This would indicate thatprobably two enzymes ore functioning in the transport of these particularamino acids. .I  -It must be emphasized that the uptake experiments are  done under con-ditions such that very little protein synthesis is occurring and remains pm- .a-partionally  constant  after irradiation.Ox  original purpose in beginning these studies was to approximate themolecular weight of the transport enzyme. Since our data indicate CImultiplicity of enzymes, we connot as yet determine individual molecularweights for the two enzymes. However, since D37 falls on  the straight .OlI I Iline portion of all the curves, we felt it would be of value to complete our322 647 1.293 I.940 2.587Dos.,ge  i n  k radscalculations for the “radiation sensitive size”  of the transport complex.Using the formula developed by Hutchinsan  and  Pollard, MW = 0.72 x lOl2  / D37, where D37 is the dose in rads  whichwill produce 37% activity, we have found a “radiation sensitive wleculw  weight” for the transport complex for phenyl-alanine of 575,000; tryptophan, 817,000; leucine, 893,000; and  glycine, 817,000.The data obtained would indicate  that the phenylolanine  transport (as well as the tryptophon and leucine systems) inNeurospora  crassa  is mediated by two  enzymes of fairly  high but  not unreasonable moleculclr  weight. The exact functionof the two  enzyme components is as yet unknown.This work was supported in part by CI  contract (AT-(40-I)-2690) between the Division of Biology and  Medicine, U. 5.Atomic Energy Commission and the Institute of Molecular  Biophysics, Florida State University. - - - Institute of MolecularBiophysics and Genetics Laboratories, Department of Biological Sciences, Florida State University, Tallahassee, Florida.

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Radiation inactivation analysis of amino acid transport systems in Neurospora crassa

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