Rates of protein synthesis in brain and other organs

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OF PROTEIN SYNTHESIS AND OTHER ORGANS

RATES

F. M.

SHAHBAZIAN.

MYRON JACOBS” and

IN BRAIN

ABEL LAJTHA

Center for Neurochemistry. The Nathan S. Kline Institute for Psychiatric Research, Ward’s Island. New York. NY I0035 and *Department of Pathobiology and Oral Pathology, College of Dentistry. New York University. New York, NY 10010. U.S.A.

Abstract-We

previously found a decrease in protein synthesis in brain during development. which was much greater as measured in brain slices than in brain i,r Go. In the present work such changes in brain were compared to those in other organs. With measurement of incorporation of flooding doses of [“Cjvaline into proteins of organs, the highest synthesis rate in the adult animal in viva was found in liver (2.2%) followed by kidney (1.8%). spleen (I .6%), lung (1.0%). heart (0.7%). brain (0.6%) and muscle (0.5%). In immature animals the synthesis rate was highest in spleen (2.6%) followed by liver (2.4%), kidney (I .7%). lung (I .6%). brain (I .S%). heart (I. 1%). and muscle (0.9%). Protein synthesis in slices from each tissue proceeded at lower rates than i~r vivu. especially in adults. The tissue affected the most by the preparation of the slices was muscle. Key words:

Protein synthesis, Organ

protein

turnover,

Slice protein

metabolism.

Development.

Rates of amino acid incorporation into proteins in the immature brain are about twice as high as in the adult organ. ” Incorporation is altered in incubated slices, with slices from immature brain incorporating amino acids at about 80% of the rate in the immature brain in Go, and slices from adult brain incorporating amino acids at about 10% of the rate of adult brain in Go. These developmental differences and the differences between in viva and in vitro incorporation are similar for most proteins and in the major portion of brain structures.4.“.‘4 In the present study we compared these alterations of protein synthesis in brain with those in other organs.

METHODS Amino

acid incorporation

in vivo

Immature and young adult Wistar rats were injected with a 500 mM [ “C]valine solution having a specific radioactivity (SRA) of 0.038 t.Xi/kmol (3 ml/100 g).” After 2 hr, the animals were decapitated and the following tissues were dissected: brain (trimmed of olfactory bulbs, cerebellum, and brain stem), median lobe of liver, kidney, spleen, heart, lung, and leg muscle. The tissues were washed with saline and chopped to 0.468-mm thickness in a McIlwain tissue slicer. To the chopped tissue 5 ml of 10% trichloroacetic acid (TCA) was added to precipitate proteins, and the tissue mixture was frozen until further use. The frozen tissue mixture was thawed, agitated in an Eberbach shaker for 15 min, and centrifuged at 6000g. The pellet was shaken with 5 ml of 10% TCA for 15 min. and centrifuged and decanted 8 times. Soluble radioactivity (free valine) in the later TRA extracts was negligible. About 5 ml of 1 N NaOH was added per gram of tissue and this mixture was incubated at room temperature for 48 hr in a stoppered tube to bring about its disintegration. The mixture was vortexed and sonicated. Aliquots of the suspension were taken for scintillation counting [tissue suspension brought to 1 ml with 1 N NaOH and 10 ml of Dimiscint (National Diagnostics)] and for protein assay.” Amino

acid incorporation

in slices

The tissues listed above were dissected, rinsed with HEPES medium, and chopped with a tissue slicer (0.468-mm thickness); about 0.5 g of tissue was incubated in 5 ml of oxygenated HEPES containing 1 mM [‘4C]valine with an SRA of 0.2 tKi/~rnol in a stoppered 25-ml Erlenmeyer flask.‘-” After 2 hr the reaction was stopped by cooling; the mixture was centrifuged in a bench centrifuge, and the pellet was washed with saline and recentrifuged. Five ml of 10% TCA was 39

F.

30

M. Shahhazim

ef trl.

added, and this mixture was vortexed and frozen until use. Further treatment ing the frozen tissue. washing it with 10% TCA several times, and determining and radioactivity as before.

consisted of thawits protein content

RESULTS The highest rates in adult tissue of amino acid incorporation irr r~it~o were observed in liver, followed in decreasing order by kidney, spleen, lung, heart, brain, and muscle (Table 1). The order of protein synthesis rates in immature organs was similar to that in adult, with liver having the highest and muscle the lowest incorporation rate; there were some deviations, such as the relatively higher rates of incorporation observed in immature spleen and brain. The ratio of rates of protein synthesis, immature-to-adult, in most tissues was almost 2: 1 except for liver and kidney, which were about 1 : 1. Comparison of rates of protein synthesis in viva with those in isirro shows that slices from ail organs exhibit rates that are lower than in the intact in viva state (Table l), just as had been shown previously for brain.” The difference is considerably greater in adult than in immature organs. For immature animals, the highest rates of synthesis in slices are found in brain, in contrast to in viva synthesis, where rates in liver are the highest. Heart and leg muscle in both immature and adult animals have the lowest rates. I.

Table

Rates of protein

synthesis in brain and other organ>

Protein synthesis, percent of total per hour IIIvilro

I/r bjit'o

-...

Immature

Immature

Adult

Adult

'!h Illl~iwolirl1'11'0 .--_-.-

Immature

Adult

_-_-_ Brain LiVU Kidney Spleen Heart Lung Leg muscle Organ samples

’’’

1.1Yto.I’~ 2.42 t 0.12 I .72 2 0.23 2.58 f 0.16 1.13~O.li 1.56iO.lX 0.93 t- O.flY

0.h2~O.O-t 2.152(1.17 1.75~0.14 1.57 k o.tir) 0.66 i 0.04 0.07+0.12 0.46 + 0.04

4

4

I .-!:!rt 0.01

ih

0.90 t O.Oh

0.t1Y7tll.ol5 0.323 t 0.070

05 27

I .K?c 0. i-1

11.2hti t O.Oil

5’,

I5

1.14+0.10 (I.35 t 0.06 I .3 i- t1.07 0.21, tr (I.03

(1.5Yh o.wi 0.162 0.022

4.t il X3 2X

38 7 4X 5

3

k t 2 -t

if. I.30 0.012 0. I70 I~.OOJ

15

3

Ratesare presented as man rt S.D. percent protein rcplaccd of total synthcsired per hour. Aver:tgcs of four experiments are given. Immature (9 days old) and young adult (6 weeks old) animals were given ;i flooding dose of [ “C~vnlinc cithcr hy i.p. injection (Ifi V;IW) or by in~u~~ti~)n (in l+rrn slice method). After 7 _ hr. the tissue was washed, disintegrated. and assayed for protein and r~~di~~etivit~ to measure the rates of protein synthesis.

DISCUSSION The rates of protein synthesis measured in this work represent averages of both rapidly and slowly metabolized protein fractions. ” Thus the percent per hour figure indicates the true amount of protein synthesized in the tissue, but this synthesis has rapid and slow components. In adult brain we estimated two compartments; the small compartment, 5%~ of the total proteins, has a 2%fold higher metabolic rate than the large (95%) compartment.‘” Since the small compartment represents only 1/20th of the proteins but its metabolism is 20 times faster, 50% of the initial incorporation occurs in this fast compartment. Even though the rate of synthesis is higher for the majority of proteins in young brain, certain proteins in the adult organ may have a higher metabolic rate. ” When rates of protein synthesis are expressed per gram of RNA, the rates in many organs are similar, except for brain, where they are somewhat lower.7h Since the amount of ribosomal RNA appears to be a good indicator of the rate of protein synthesis, and since most of RNA is ribosomal, in viva differences in rates of protein synthesis probably reflect differences in concentration of RNA. Dunlop et al.” have shown that the rate of protein synthesis is higher in the immature than in the adult brain because of a greater number of ribosomes rather than a greater

Protein synthesis rates in organs

41

RNA activity. These investigators showed that the mg of protein synthesized per hour expressed per mg of RNA was identical in the brains of immature and adult rats. Most of the research on rates of protein synthesis in organs has been done on adult animals.’ The findings on protein synthesis rates in adult organs in vivo and in vitro presented in this study are in general agreement with such data when available in the literature.“‘~‘~~‘X~20-22 Most immature organs show rates of protein synthesis that are about twice that of adult, except for kidney and liver. For brain, it was found that breakdown is also higher in the immature organ, hence only a part of the higher rate of protein synthesis in the immature (as compared to the adult) results in growth. The increased breakdown may be the result of cell death and protein conversion.2 The high rate of breakdown in young also shows that in most immature tissues the protein synthesis rate is greatly in excess of that required for growth, and growth can be controlled by alteration of breakdown rates as well as of protein synthesis. It is reported that adult organs such as heart, lung, spleen, liver, kidney, and brain under conditions of stress synthesize a ‘stress protein’, which constitutes the main portion of the newly formed protein in the soluble fraction.“*‘* One of the major ‘stresses’ invariably induced when organ slices are prepared for incubation is the physical trauma of sectioning. The protein produced under such in vitro conditions could be the same as the ‘stress protein’ induced in the living animal. Stress proteins have been thought to represent the greater part of the protein synthesized by organs 30 min after trauma. In general, stress proteins appear to be synthesized in greater amounts in adult organs than in immature ones, and in certain organs such as brain, immature animals appear not yet to synthesize stress proteins.27 As our results in fractionation of proteins according to mol. wt of subunits showed,” stress proteins are not the major proteins synthesized in slices of brain tissue under our experimental conditions. This also appears to be a possible conclusion from other work reported by Cosgrove and Brown.’ In each case, with immature tissue incorporation in slices was closer to in viva incorporation than with adult tissue. In brain this developmental difference was noted for each region and cell type, and the synthesis of most proteins was affected to a greater degree in slices of adult tissue.2J.2s The reason for this greater post-mortem alteration of protein synthesis in adult as compared to immature tissue is not clear at the present time. A comparison of data in the literature indicates that incorporation of amino acids during tissue perfusion occurs at a higher rate than in slices and in some tissues it is close to in viva synthesis rates. In any study of alterations of protein metabolism, differences among various systems used for incorporation and the changes in such differences during development have to be taken into consideration.

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