Polyphosphate:AMP phosphotransferase and polyphosphate:ADP phosphotransferase activities of Pseudomonas aeruginosa

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is <75.     

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.     

Polyphosphate glucokinase from Propionibacterium shermanii. Kinetics and demonstration that the mechanism involves both processive and nonprocessive type reactions

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose.     

Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation, and environmental stress in Trypanosoma cruzi

Inorganic polyphosphate (polyP) has been identified and measured in different stages of Trypanosoma cruzi. Millimolar levels (in terms of P(i) residues) in chains of less than 50 residues long, and micromolar levels in chains of about 700--800 residues long, were found in different stages of T. cruzi. Analysis of purified T. cruzi acidocalcisomes indicated that polyPs were preferentially located in these organelles. This was confirmed by visualization of polyPs in the acidocalcisomes using 4',6-diamidino-2-phenylindole. A rapid increase (within 2--4 h) in the levels of short and long chain polyPs was detected during trypomastigote to amastigote differentiation and during the lag phase of growth of epimastigotes (within 12--24 h). Levels rapidly decreased after the epimastigotes resumed growth. Short and long chain polyP levels rapidly decreased upon exposure of epimastigotes to hypo-osmotic or alkaline stresses, whereas levels increased after hyperosmotic stress. Ca(2+) release from acidocalcisomes by a combination of ionophores (ionomycin and nigericin) was associated with the hydrolysis of short and long chain polyPs. In agreement with these results, acidocalcisomes were shown to contain polyphosphate kinase and exopolyphosphatase activities. Together, these results suggest a critical role for these organelles in the adaptation of the parasite to environmental changes.     

The mechanism of utilization of polyphosphate by polyphosphate glucokinase from Propionibacterium shermanii

The phosphorylation of glucose by polyphosphate glucokinase with both long- and short-chain polyphosphates has been shown to occur by either a nonprocessive mechanism, i.e. with repeated association and dissociation of the polyphosphate from the enzyme after each phosphorylation or by a quasiprocessive mechanism in which several phosphorylations occur prior to the release of polyphosphate and the reassociation with the enzyme. In contrast, the phosphorylation of ADP to ATP by polyphosphate kinase is by a strictly processive mechanism; the phosphorylation occurs without release of the polymer from the enzyme prior to termination of the reaction (Robinson, N. A., Clark, J. E., and Wood, H. G. (1987) J. Biol. Chem. 262, 5216-5222). The demonstration that the mechanism is quasi-or nonprocessive was accomplished by electrophoresis using a variety of concentrations of polyacrylamide gels which made it possible to detect the intermediate sizes formed during the reactions. It also has been shown that all chains longer than about 100 are used simultaneously, but with chains of less than 100 residues, there is preferential utilization of the longest chains. Thus a narrow range of sizes is formed from a heterogeneous mixture of long chains. It is this formation of the narrow range of sizes that makes it possible to use polyphosphate glucokinase for the determination of the average size of long chains (Pepin, C. A., Wood, H. G., and Robinson, N. A. (1986) Biochem. Int. 12, 111-123).     

Different chain length specificity among three polyphosphate quantification methods

Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbuscular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which polyphosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Polyphosphate kinase from Propionibacterium shermanii: formation of an enzymatically active insoluble complex with basic proteins and characterization of synthesized polyphosphate

Polyphosphate kinase, which catalyzes the synthesis of polyphosphate from ATP, has been partially purified from Propionibacterium shermanii. The reaction is unusual in that addition of basic protein causes the enzyme to precipitate and the insoluble form has optimal activity. The synthesized [32P]polyphosphate is non-covalently bound to the precipitated material and was isolated from the complex by proteolysis. The gel electrophoresis procedure of Maxam and Gilbert was adapted to sizing polyphosphates. When polyphosphate was treated with alkali, polyphosphates ranging from 1-100 phosphate residues were obtained as individual bands. The untreated enzymatically synthesized polyphosphate migrated as a species in excess of 200 phosphate moieties.     

Primary structure of inorganic polyphosphate/ATP-NAD kinase from Micrococcus flavus,and occurrence of substrate inorganic polyphosphate for the enzyme

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.     

Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.     

Polyphosphate-Mediated Inhibition of Tartrate-Resistant Acid Phosphatase and Suppression of Bone Resorption of Osteoclasts

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α’-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.     

Studies of the DNA helicase-RNA primase unit from bacteriophage T4. A trinucleotide sequence on the DNA template starts RNA primer synthesis

The purified DNA replication proteins encoded by genes 41 and 61 of bacteriophage T4 catalyze efficient RNA primer synthesis on a single-stranded DNA template. In the presence of additional T4 replication proteins, we demonstrate that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for RNA primer-dependent initiation of new DNA chains. These chains start with primers that have the sequences pppApCpNpNpN and pppGpCpNpNpN, where N can be any one of the four ribonucleotides. Each primer is initiated from the T (A-start primers) or C (G-start primers) in the center of the recognized template sequence. A subset of the DNA chain starts is observed when one of the four ribonucleoside triphosphates used as the substrates for primer synthesis is omitted; the starts observed reveal that both pentaribonucleotide and tetraribonucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive. It was known previously that, when 61 protein is present in catalytic amounts, the 41 and 61 proteins are both required for observing RNA primer synthesis. However, by raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein. The DNA chains made are initiated by primers that seem to be identical to those made when both 41 and 61 proteins are present; however, only those template sites containing the 5'-GCT-3' sequence are utilized. The 61 protein is, therefore, the RNA primase, whereas the 41 protein should be viewed as a DNA helicase that is required (presumably via a 41/61 complex) for efficient primase recognition of both the 5'-GCT-3' and 5'-GTT-3' DNA template sequences.     

Introduction of polyphosphate as a novel phosphate pool in the chloroplast of transgenic potato plants modifies carbohydrate partitioning

Potato plants (Solanum tuberosum L., cv. Désirée) were transformed with the polyphosphate kinase gene from Escherichia coli fused to the leader sequence of the ferredoxin oxidoreductase gene (FNR) from Spinacea oleracea under the control of the leaf specific St-LS1 promoter to introduce a novel phosphate pool in the chloroplasts of green tissues. Transgenic plants (cpPPK) in tissue culture developed necrotic lesions in older leaves and showed earlier leaf senescence while greenhouse plants showed no noticeable phenotype. Leaves of cpPPK plants contained less starch but higher concentrations of soluble sugars. The presence of polyphosphate in cpPPK leaves was demonstrated by toluidine blue staining and unambiguously verified and quantified by in vitro 31P-NMR of extracts. Polyphosphate accumulated during leaf development from 0.06 in juvenile leaves to 0.83 mg P g-1 DW in old leaves and had an average chain length of 18 residues in mature leaves. In situ 31P-NMR on small leaf pieces perfused with well-oxygenated medium showed only 0.036 mg P g-1 DW polyphosphate that was, however, greatly increased upon treatment with 50 mM ammonium sulfate at pH 7.3. This phenomenon along with a yield of 0.47 mg P g-1 DW polyphosphate from an extract of the same leaf material suggests that 93% of the polyphosphate pool is immobile. This conclusion is substantiated by the observation that no differences in polyphosphate pool sizes could be discerned between darkened and illuminated leaves, leaves treated with methylviologen or anaerobis and control leaves, treatments causing a change in the pool of ATP available for polyPi synthesis. Results are discussed in the context of the chelating properties of polyphosphates for cations and its consequences for the partitioning of photoassimilate between starch and soluble sugars.     

Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.     

Interaction between the synthesis of alpha and beta globin.

We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.