Héctor S. Barra

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE‐SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE‐INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G‐25‐40 was able, after addition of some factors, to incorporate [I4C]arginine (apparent Km= 5 μM) and [14C]tyrosine (apparent Km= 20 μM) into its own proteins. The factors required for the incorporation of [14C]arginine were: ATP (optimal concentration = 0‐25‐2 μM) and Mg2+ (optimal concentration 5 mM). For the incorporation of [I4C]tyrosine the required factors were: ATP (apparent Km= 0‐75 μM), Mg2+ (optimalconcentration 8‐16 mM) and K+ (apparent Km= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [14C]arginine and [14C]tyrosine, respectively, 7‐4 and 7‐0 in phosphate buffer and 7–9 and 7‐3‐8‐1 in tris‐HCl buffer. Pancreatic ribonuclease abolished the incorporation of [14C]arginine but had practically no effect in the incorporation of [14C]tyrosine. Furthermore, [14C]arginyl‐tRNA was a more effective donor of arginyl groups than [14C]arginine, whereas [14C]tyrosyl‐tRNA was considerably less effective than [14C]tyrosine. The incorporations of [14C]arginine and [14C]tyrosine into brain proteins were from 25‐ to 2000‐fold higher than for any other amino acid tested (12 in total). In brain [14C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [14C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [14C]Arginine was bound so that its a‐amino group remained free. Maximal incorporation of [14C]tyrosine in brain of 30‐day‐old rats was about one‐third of that in the 5‐day‐old rat. The changes with postnatal age in the incorporation of [14C]arginine were not statistically significant.

Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins.

Abstract Properties so far studied of the protein that incorporates tyrosine show remarkable similarities with those of the microtubule proteins. The molecular weight of proteinyl-14C-tyrosine determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 54,000. The acceptor protein and proteinyl-14C-tyrosine were found in different states of aggregation; one of these states is apparently a dimer of molecular weight approximately 110,000. From a single preparation of proteinyl-14C-tyrosine variable proportions of dimer and higher molecular weight aggregates were obtained by incubating in different conditions. Proteinyl-14C-tyrosine was eluted from DEAE-Sephadex A-50 similarly to 3H-colchicine-tubulin complex. The pattern of elution from Sephadex G-200 of dimer proteinyl-14C-tyrosine was similar to that of 3H-colchicine-tubulin complex. Proteinyl-14C-tyrosine was precipitated with vinblastine sulfate.

Posttranslational tyrosination/detyrosination of tubulin

Tubulin can be posttranslationally modified at the carboxyl terminus of the α-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of α-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of α-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.

Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase.

SummaryThe carboxypeptidase previously described3 that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mm MgCl2 or 30 mm KCl. Mercaptoethanol (50 mm), colchicine (0.2 mm) and tyrosine (0.2 mm) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mm), deoxycholate (0.5%), CuCl2 (0.1 mm), ZnC12 (0.1 mm) and NaCl or KCI (240 mm) had a strong inhibitory effect. The optimal pH of this enzyme. was 6.3–7.A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg2+, K− and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl2 was 3–20 mm and of KCl was 10–30 mm. For ADP the maximal activity was at 0.3 mm or higher.An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not.

RELEASE OF TYROSINE FROM TYROSINATED TUBULIN. SOME COMMON FACTORS THAT AFFECT THIS PROCESS AND THE ASSEMBLY OF TUBULIN

An enzyme system present in the soluble fraction of rat brain homogenate catalyzes the incorporation of tyrosine into the carboxyl end of the a-subunit of tubulin [ 1,2] . The system does not require nucleic acids [3]. Results from studies on the incorporation of [r4C] tyrosine into brain tubulin in animals whose protein synthesis was inhibited by cycloheximide indicated that a similar system operates in vivo [4]. The present work deals with an activity found in the soluble fraction of rat brain that determines the release of the C-terminal tyrosine from tyrosinated tubulin. Every previously known inhibitor or activator of assembly of tubulin we tested also affected the release of tyrosine when assayed in similar conditions. The relationship of the activity reported here with a similar one previously described [S] for which was claimed that ADP and Pi were required is discussed.

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [14C]tyrosine into the C‐terminal position of α‐tubulin of rat brain cytosol was 10‐fold higher for non‐assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non‐assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.

Tubulinyl-tyrosine Carboxypeptidase from Chicken Brain: Properties and Partial Purification

Abstract: Tyrosine can be released from tubulinyl‐tyrosine by the action of a brain carboxypeptidase. The molecular weight of this enzyme found by gel filtration through a column of Sephadex G‐200 was 90,000. The enzyme was very unstable in a purified preparation in which the activity per milligram of protein was increased 250‐fold with respect to the starting material. The precise magnitude of the purification cannot be stated because of the unknown amount of endogenous tubulinyl‐tyrosine in the material to be assayed. A comparative study was done between tubulinyl‐tyrosine carboxypeptidase (TTCP) activity and pancreatic carboxypeptidase A (CPA, EC 3.4.12.2) activity using tubulinyl‐[14C]tyrosine as substrate. The most remarkable differences found are: MgCl2 (2 mM), phenyl acetate (10 mM), or EDTA (5 mM) increased the TTCP activity whereas the CPA activity was strongly inhibited by these compounds, lodoacetate (2 mM) and ZnCl2 (0.1 mM) inhibited the TTCP activity more than the CPA activity. Contrarily, mercaptoethanol (50 mM) and dimethyl sulfoxide (5%) showed a stronger inhibitory effect on CPA than on TTCP. Of several N‐carbobenzoxy dipeptides (Z‐dipeptides) tested the greatest inhibitory effects on TTCP activity were obtained with Z‐Glu‐Tyr and Z‐Glu‐Phe, although strong inhibitory effects on CPA were also obtained with other Z‐dipeptides.

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot‐TCA‐insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase‐insensitive and required ATP (apparent Km= 0.64 mm), KC1 (apparent Km= 14 mm) and MgCl2 (optimal concentration range 4‐15 mm). The apparent Km for phenylalanine was 2.9 mm. [14C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [14C]phenylalanine and [14C]tyrosine when incubated simultaneously or successively. The Km and Kt (3.3 mm) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [14C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [14C]Phenylalanine and [14C]tyrosine entered into COOH‐terminal positions in the reactions described. Brain exhibited a 25‐ to 100‐fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin☆

In vitro incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [14C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [14C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [14C]tyrosine. The α and β subunits of tubulin from animals that received [14C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of αβ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [14C]amino acids was injected, the radioactivity ratio of αβ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.

Tubulin must be acetylated in order to form a complex with membrane Na + ,K + -ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na+,K+-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na+,K+-ATPase and consequent inhibition of enzyme activity.