Carrier-mediated Uptake of Arginine and Urea by Volvox carteri

Volvox carteri f. naganensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but It is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The Ki values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective Km values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V. values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program. The use of mixed, radioactive amino acids to study protein synthesis in the simple, autotrophic, multicellular eukaryote, Volvox carteri f. nagariensis, led to an unexpected observation: in this species arginine is taken up from the external medium and utilized in protein synthesis far more rapidly than any ofthe other common amino acids of proteins, including lysine (7). This led to the speculation (confirmed by preliminary observations) that Volvox possesses only one carrier capable ofmediating amino acid uptake, and that this carrier is arginine-specific. If verified, this would appear to be a novel situation. Some algae have been shown to accumulate arginine more rapidly than neutral or acidic amino acids (6, 11, 12), yet saturable carriers for the latter two classes of amino acids have usually been detected in other species, at least in nitrogen-starved cultures. The first objective of the work presented here was to reexamine the selectivity of amino acid uptake by Volvox under a variety ofculture conditions, including nitrogen starvation. We found no evidence for any amino acid carrier other than an arginine-specific one under any culture conditions; but in the course of these studies, we determined that arginine is incapable of supporting growth of Volvox when supplied as the only source of nitrogen in the medium. What is the adaptive significance of being able to accumulate rapidly one, and only one, amino acid when that amino acid cannot be utilized as a sole nitrogen source? In pondering that question we were led to the speculation that the carrier involved in the arginine uptake might have physiological significance because of its capacity to transport urea, and that arginine accumulation might be merely a trivial, secondary consequence of imperfect structural specificity of the urea carrier. Urea has been shown to serve as an efficient nitrogen source for a wide variety of algae (14, 15) and in several algae (including a close relative of Volvox, Chlamydomonas) it has been demonstrated that urea is taken up by a saturable, carrier-mediated process (3, 10, 16). Furthermore, Volvox flourishes in polluted farm ponds where the capacity to transport and utilize urea as a nitrogen source should be of selective value. Therefore, the second objective of the work presented here was to examine urea uptake by Volvox under a variety of culture conditions and to ascertain (by quantitative and qualitative examination of the kinetic properties and specificities of the arginine and urea uptake systems) whether urea and arginine are taken up by the same carrier in Volvox. We find that they are not. Finally we report the pattern of temporal variation observed in the rates of arginine and urea uptake throughout the asexual life cycle of synchronous Volvox cultures. MATERIALS AND METHODS Axenic cultures of V. carteri f. nagariensis, strain HK10 (originally obtained from the Indiana University Culture Collection of Algae, now the Culture Collection of Algae at the University of Texas), maintained in the asexual state, were used throughout this study. Standard growth conditions (including the use of medium containing 0.5 mM Ca(NO3)2, where not otherwise indicated) were as previously reported (7) except for four minor changes, all of which have been found to improve the degree of developmental synchrony: (a) the standard Volvox medium is now routinely supplemented with 20 mg ofNa2CO3/l; (b) the temperature during the illumination period has been raised to 32 C; (c) each 48-hr cycle now comprises 32.5 hr of light followed by 15.5 hr of darkness; and (d) the level of illumination (provided by cool white fluorescent lights) has been increased to 215 ILE/m2 sec (measured with a Lambda Instrument Company Li-Cor model Li-185 meter with a quantum-type sensor; this corresponds to 1,500 ft-c, measured as previously reported (1)). Under these conditions Volvox develop synchronously and complete one asexual life cycle each 48 hr. Transfers of aerated stock cultures were routinely made every 48 hr, 24 hr after the beginning of each illumination period. Except in the experiments where uptake was studied throughout the course of a 48-hr culture period, uptake studies were performed between the 3rd and 8th hr of the illumination period using cultures that contained 100 to 300 Volvox spheroids/ml. All incubations with radioactively labeled substances were performed in sterile, loosely capped (but unaerated) culture tubes (13 x 100 mm) in a 32 C water bath. Concentrations of 3H precursors were selected arbitrarily to provide maximum sensitivity, but concentrations of 14C precursors were chosen to be in the range of the corresponding Km values and concentrations of inhibitors were chosen on the basis of preliminary estimates of the corresponding values for Ki. All incubations were carried out at an illumination level of 215 ,4E/m2 sec, except that those performed in the normally dark period were carried out in a room illuminated only by a low wattage "night light." At predetermined intervals, 5-ml 549 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 61, 1978 aliquots were withdrawn under axenic conditions from 500-ml bubbler-flask cultures, added to appropriate tubes, mixed by inversion, and transferred to the water bath within 7 sec. At the end of the desired incubation period, organisms were rapidly collected and washed by vacuum filtration on nitrocellulose filters; filters were frozen at once on dry ice and stored dry, at -20 C, until the time of analysis. At each time point at least two equivalent tubes were incubated, experiments were performed at least twice (in different weeks or months), and all subsequent analyses were performed in duplicate. Hence, each data point represents the average of at least eight (and often more) determinations of each relevant parameter. In all prolonged experiments samples were removed from the culture flask at intervals during the experimental period and fixed with glutaraldehyde; these samples were subsequently examined microscopically to determine culture density and degree of developmental synchrony throughout the culture period. At the time of analysis organisms were removed from the filters and disrupted with ultrasound in 1.3 ml of ice-cold standard Volvox medium as previously described (7). Duplicate 0.2-ml aliquots were removed from each sonicate for determination of protein concentration by the method of Lowry et al. (9). Quadruplicate 0.15-ml aliquots were transferred to Whatman GF/A filter discs; two of these were used to determine total radioactivity and two to determine the level of trichloroacetic acid-insoluble radioactivity; TCA'-soluble radioactivity was determined by difference. Routinely the TCA extraction was performed in the cold, but occasional samples were subjected to both cold and hot TCA extractions. Radioactivity was measured in a liquid scintillation spectrometer, counting efficiency was determined for each sample as previously described (7), and observed counting rates were converted to disintegration rates. Disintegration rates were converted to molar values using specific radioactivity data supplied by the vendor of the labeled substance. Chromatography was used to analyze the chemical nature of the TCA-soluble radioactivity in [3HJarginine-labeled cultures. TCA extracts of organisms cultured for 1 hr in the presence of [3Hlarginine were prepared. These extracts were then chromatographed on silica gel TLC plates (E. Merck, Co.) in parallel with chromatographic standards of arginine, citrulline, ornithine, lysine, histidine, arginosuccinic acid, and the neutral and acidic amino acids commonly found in proteins. The plates were developed in a 1:1 mixture of l-propanol and 28% NH40H (which separates arginine from all other common amino acids and all known arginine metabolites). Following ninhydrin treatment of the plate to identify location of arginine and all other standards, the silica gel layer was subdivided into multiple narrow zones and each zone was scraped off the glass backing and transferred to scintillation vials for counting. Intracellular concentrations of radioactive substances were estimated by multiplying radioactivity/mg of cellular protein by 33.3 mg of protein/ml; this factor relating protein content to packed cell volumes had been previously obtained for Volvox cells (mixed somatic and reproductive cells) that had been isolated by a modification of the method previously described (7). Mixed L-3H-amino-acids (15 Ci/g), L-[3-3Hlarginine (10.2 or 13 Ci/mmol), L-[4,5-3Hlleucine (60 Ci/mmol), L-[4,5-9HJlysine (63.9 Ci/mmol), and L-[3,4-3H]glutamate (45 Ci/mmol) were obtained from New England Nuclear Corporation. L-[U-'4C]arginine (240 or 270 mCi/mmol) and [U-_4C]urea (51.2 mCi/mmol) were obtained from ICN Pharmaceuticals. Structural analogs used were L-argmine, D-arginine, L-canavanine, L-ornithine, L-citrulhne, L-lysine, urea, hydroxyurea, guanidine hydrochloride, and methylamine from Sigma Chemical Co., L-homoarginine from ICN Pharmaceuticals, and ammonium chloride and potassium cyanate from Baker Chemical Co. Dowex 1 was obtained from Sigma Chemical Co. and prepared for use by washing with base, acid, and copious amounts of water, according to standard procedures. Regression analyses and statistical evaluations were performed on a programmable hand calculator (Hewlett-Packard HP-25).