Reversible Inhibition by Histidinol of Protein Synthesis in Human Cells at the Activation of Histidine*

Abstract l-Histidinol has been found to be a potent, reversible inhibitor of protein synthesis in cultured human cells. When the culture medium contains 0.005 mm histidine, protein synthesis is inhibited 50% by 0.1 mm histidinol. Histidinol does not significantly affect the transport of histidine into cells or the amount of histidine accumulated in the free amino acid pool. Histidinol probably inhibits protein synthesis by decreasing the activation of histidine since it competitively inhibits the pyrophosphate-ATP exchange reaction promoted by crude histidyl-tRNA synthetase in the presence of histidine, with an apparent Ki of 4 x 10-7 m. Histidinol alone will not support the exchange reaction. The inhibition by histidinol of the over-all histidine charging reaction with tRNA is also competitive. The apparent Ki of histidinol for the charging reaction, under different reaction conditions than the exchange reaction, is 3 x 10-6 m. Histidinol appears to be a useful inhibitor for studying the regulation of macromolecule synthesis in cultured human cells.

has been found to be a potent, reversible inhibitor of protein synthesis in cultured human cells.
When the culture medium contains 0.005 fll~ histidine, protein synthesis is inhibited 50% by 0.1 mM histidinol. Histidinol does not significantly affect the transport of histidine into cells or the amount of histidine accumulated in the free amino acid pool.
Histidinol probably inhibits protein synthesis by decreasing the activation of histidine since it competitively inhibits the pyrophosphate-ATP exchange reaction promoted by crude histidyl-tRNA synthetase in the presence of histidine, with an apparent Ki of 4 X lo-' M. Histidinol alone will not support the exchange reaction.
The inhibition by histidinol of the over-all histidine charging reaction with tRNA is also competitive.
The apparent K; of histidinol for the charging reaction, under different reaction conditions than the exchange reaction, is 3 x 10v6 M. Histidinol appears to be a useful inhibitor for studying the regulation of macromolecule synthesis in cultured human cells.
studies of the regulation of macromolecular synthesis in cultured mammalian cells currently employ a variety of inhibitors of nucleic acid and protein synthesis.
Two compounds, 5-methyltryptophan (1) and 0-methylthreonine (2), have been shown to competitively inhibit, respectively, the activation of tryptophan and isoleucine by the corresponding aminoacyl synthetases of mammalian cells. It has been demonstrated that O-methylthreonine is a reversible inhibitor of protein synthesis in intact mammalian cells (3)(4)(5), while we have found that B-methyltryptophan is not an effective inhibitor of protein synthesis in intact HeLa cells, presumably because of failure to enter the cells. In searching for additional competitive inhibitors of activation of amino acids essential to human cells, which would not be charged onto tRNA, we have found that histidinol, a normal precursor of histidine iu procaryotes and some eucaryotes, * This investigation was supported by a research grant from the National Institutes of Health (CA-10922).
$ Career Development Awardee of the United States Public Health Service, National Institutes of Health (K4-GM-lG,281). competitively inhibits histidine activation in HeLa cells. While histidinol has not previously been observed to be an inhibitor of protein synthesis, others have showu that histidinol (6) and the amino alcohols derived from methionine (7), tyrosine (8), isoleutine (7), and leucine (9) competitively inhibit in vitro the activation of these amino acids by the nminoacyl synthetases of bacteria. [14C]Leucine (New England Nuclear Corp.) was added 10 min later to each culture, to a final specific activity of 9.6 mCi per mmole.
Duplicate 0.5-ml samples were removed at intervals and processed for determination of "C incorporation into protein as previously described (11,12 Co. PR-6 centrifuge, as previously described (14). Mitochondria and membranes were removed by centrifugation for 15 min at 10,000 rpm in the Sorvdl RC-213 centrifuge.
The supernatant was then centrifuged for 45 min at 50,000 rpm in the A321 rotor in the Internat,ional Equipment Co. I3 60 centrifuge, and the entire supernatant down to the ribosomal pellet was removed.
This supernatant was applied to a DEAE-cellulose column, 1.5 x 6 cm, equilibrated with 20 mM Tris, pH 7.6, 2 fnM MgC&, 1 mM dithiothreitol, 0.1 mM EDTA. The column was rinsed with 40 ml of the equilibration buffer and was then eluted with this buffer with NaCl raised to 0.3 at. The elution of histidine-activating enzyme was followed by the exchange of [32P]pyrophosphate into ATP as described below. The active material was pooled and precipitated with (NH&SOI at 50% of saturation. The precipitate was dissolved in a minimal amount of 0.1 InM dithiothreitol, 2 mM MgClz, 150/, glycerol, pH 7.4, and was desalted and cleansed of free amino acids by passage through a column of coarse Sephadex G-25, 1.3 x 31 cm, equilibrated with this solution. Active fractions were pooled and stored at -70".
All operations were at 4".
The sample was filtered onto a Millipore filter, rinsed 20 times with 3 ml of cold water, and dried for counting with a gas flow counter. Blank incorporation was determined from reaction mixtures with enzyme, without addition of histidine.
Incorporation was linear for at least 20 min and did not exceed 176 of the added Initial experiments showed that under the conditions described the reaction had a magnesium concentration optimum around 20 mM; no exchange occurred below 3 rnbf. Under the conditions adopted for the assay the apparent K, values, at 1 mM histidine, were found to be 1.3 X lOma M for ATP and 1.4 x 10-S M for pyrophosphate.
Determination of IIistidine Binding to tRNA-Transfer RNA was isolated from whole HeLa cells or their cytoplasmic extract by a hot phenol-sodium dodecyl sulfate procedure at 60' as previously described (15). After ethanol precipitation the RNA was dissolved in 0.1 M NaCl, 0.01 M Tris, 0.01 M EDTA. 0.5% sodium dodecyl sulfate, pH 7.4, and sedimented for 17 hours at 26,000 rpm on 15 to 30% sucrose gradients, in the latt.er buffer, in the SW 40 rotor of the Beckman centrifuge.
The ult,raviolet absorption profile of the gradients was monitored, and the peak containing tRNA was collected and precipitated with ethanol.
The assay mixture (modified from Reference 16) for histidine binding contained in 100 ~1: 2.5 mM ATP; 0.25 mM CTP; [14C]histidine (278 mCi per mmole) and histidinol as specified; the other 19 amino acids at 0.013 mM each, nonradioactive; 0.32 As0 unit of tRNA, and crude enzyme, 0.024 mg. Reactions were initiat,ed by addition of enzyme to reaction mixture previously incubated to 37". Aliquots of 20 ~1 were applied to linch filter paper discs (Whatman 540), according to the method of Bollum (17), and were immediately placed in 107, trichloroacetic acid containing 1 mg per ml of nonradioactive histidine. The filters were rinsed twice through 5% trichloroacetic acid, once through 95% ethanol, and were dried for counting in a toluene-based scintillator fluid (Omnifluor, New England Nuclear Corp.) in a Packard counter.
Since the observed inhibitions of ATP-pyrophosphate ex-change and of histidine charging by histidinol are competitive, as judged from Lineweaver-Burk p1ot.s of initial velocities versus substrate concentration, the values of apparent K i were determined from plots of the concentration of histidinol versus the reciprocals of initial velocities (18).

Inhibition
of Protein Synthesis by Histidinol-As shown in Fig. 1, histidinol is an inhibitor of protein synthesis in HeLa cells. The inhibition requires some 30 min to become maximal, presumably due to a relatively slow entry of histidinol into the cells. Addition of excess histidine rapidly and completely restores the normal rate of protein synthesis, as shown in Fig. 1. The dependence of the inhibition on histidinol concentration is shown in Fig. 2.
,4s indicated in Table I histidinol has no significant effect on either the rapid transport of histidine into HeLa cells, as shown by entry into the acid-soluble pool during a 2-min incubation with labeled histidine, or the level of free histidine in the cells, as shown by incorporation into that pool after a 24.min incubation. This result excludes the possibility that t'he observed inhibition of protein synthesis is due to a histidinol-mediated starvation of the cells for histidine.
Inhibition by Histidinol of Pyrophosphate-ATP Exchange Promoted h Hisfidine--The possibility that histidinol may interfere with histidine activation was tested by examining its effects on histidine-promoted exchange of [Vlpyrophosphate into ;1TP. The incorporation of n-[14C]leucine into the total protein of HeLa cells was determined as described under "Experimental Procedure." Three identical cultures were used: A, control culture; B, L-histidinol (0.4 mn) was added 10 min prior to addition of [r%]leucine; C, L-hist,idinol was added as in B, and then additional histidine was added to 1 rnn at the time indicated by the arrow. All cultures initially contained 0.005 mM histidine. into acicLsolu6le pool of HeLa cells, with and without L-histidinol Cells at a concentration of 2 X 10b per ml were incubat,ed 10 min at 37" in complete medium containing0.005 mM hi&dine, wit.h and without 0.2 mM L-histidinol.
L-[l%]Histidine (45 mCi per mmole, final specific activity) was then added, and 2-ml samples of cells were t,aken 2 and 24 min later for centrifugation and rinsing with ice-cold Earle's salts solution.
The free amino acids were extracted with cold 169; trichloroacetic acid from the cell pellets, and t.he insoluble debris was removed by centrifugation. After ext.raction withether to remove trichloroacetic acid aliquots of the supernatant solrttion were taken for determination of radioactivit,y by scintillation counting. The double reciprocal plot of velocities versus histidine concentration at differing histidinol concentrations, Fig. 4, indicates that histidinol is a caompetitive inhibitor of histidine activation.
The apparent K, for histidine is 1.0 X 10e5 M for the exchange reaction and apparent Ki for histidinol is 4 X lo-' M.
Inhibition by Histidinol of Uistidine Charging onto tRN,L-111 view of t,he competitive inhibition of histidine-promoted pyrophosphat,e exchange by histidinol, this compound would be experted to competitively inhibit the charging of tRNA wibh histidine.
As shown in Fig. 5 this is the case. The addition of histidinol to charging assays was also found not to affect the final level of charging of tRNA with histidine.
The apparent. Km for histidine for charging was 7 X 10e6 M and apparent Ki for histidinol was 3 X 10e6 M. It is interesting to note that although the Km for histidine is similar for both pyrophosphate eschange and the over-all charging reaction, the K i of histidinol is 8 times lower for the exchange than for the over-all reaction.
Since t'he enzyme used was not purified, the significance of this observation is at present unclear. DISCUSSIOK Our results establish that histidinol is a potent, reversible inhibitor of protein synthesis in human cells. The data indicate that histidinol acts at the level of histidine charging to tRNA by competitively inhibiting histidine activation. The shape of the dose-inhibition dependence curve for protein synthesis inhibition and the prompt, full reversal of the inhibition by excess histidine are consistent with this explanation.
The fact that histidinol takes some time to exert maximal inhibition of protein synthesis in intact cells is probably due to a comparatively slow entry of the inhibitor into the cell. We have not studied histidinol transport, but we have observed that the compound does not appear to significantly interfere with histidine transport into the cell.
The observation that histidinol ail1 not support pyrophosphat,e-ATP exchange and does not affect the final level of histidine charging effectively rules out the possibility that histidinol is activated.
There is thus no reason to suppose that histidinol interferes with the charging capacity of the histidine acceptor tRNA species. Since mammals cannot carry out the de novo synt,hesis of histidine, histidinol probably does not occur in human cells. However, histidinol is synthesized during histi-dine formation in the procaryotes and lower eucaryotes (19,20), and it has been reported (6) that it competitively inhibits the activation of histidine by Salmonella typhimurium histidyl-tRNA synthetase, with a K; of 5 X lop5 hr.
Another competitive inhibitor of amino acid activation, Omethylthreonine (2), which competes with isoleucine, has been used in several studies of protein synthesis and its regulation in mammalian cells (4,5,21). Histidinol and 0-methylthreonine are useful compounds for the study of regulation of macromolecule synthesis in cultured human cells because they are effective inhibitors at low concentrations with intact cells. Their effects are rapidly, specifically reversible by the corresponding amino acid and neither compound is attached to tRNA.
Both of these inhibitors can be used to rapidly put cultured cells into a condition similar to that achieved by depriving the cells for the corresponding amino acid for several hours (2,12). Furthermore, because of endogenous protein turnover, the decrease in the absolute rate of protein synthesis obtained by amino acid deprivation is limited to about 80% (la), while these inhibitors can virtually abolish protein synthesis at high concentrations relative to the amino acids with which they compete.