R. Caputto

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.

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.

Studies of brain weight and RNA content after short periods of exposure to environmental complexity.

Abstract Rats in Environmental Complexity (EC) were compared with litter mates maintained in Impoverished Conditions (IC) for various times. An initial relatively fast onset of the difference between the cerebral weight of the EC and IC rats was observed. This phase was followed by another in which this difference was not statistically significant. The third period was characterized by a slow increase of the difference in cerebral weight of the EC and IC rats. The RNA content per gram of wet tissue was higher in the EC than in the IC animals in the period of fast acquisition of difference in brain weight, but in the successive periods (60 days in total) no definite indications of differences in the RNA/gram of wet tissue were found. The differences of RNA content per cerebrum followed a pattern similar to the differences of cerebral weight. Body and hypophysis weights were higher in IC than in EC animals after 2 days in their respective conditions and throughout the rest of the experiment.

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.

Biosynthesis of Brain Gangliosides

The pathways of synthesis of brain gangliosides have been inferred from results of usual enzymic experiments in which all the substrates are from exogenous origen and from results of experiments in which endogenous acceptors present in subcellular membranes are labelled by using exogenous, water soluble donors. For the synthesis of most gangliosides the results obtained with both methods are in agreement but in the synthesis of GDIb they are discrepant. The pathways obtained with each method are summarized in Fig. 1a and 1b. In the present communication we will deal preferentially with progress made in this field since the ganglioside conference at Strasbourg in 1973 (see ref. 12).

THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM

Abstract– In the retinas of 1‐day‐old chickens that received an intraocular injection of N‐[3H]acetylmannosamine the labelling of N‐acetylneuraminic acid and CMP‐N‐acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20‐fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N‐[3H]acetylmannosamine was injected intraocularly, but when N‐[3H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N‐[3H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10‐fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.

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.