Albumin and transferrin synthesis during development in the rat

In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin.     

Effect of corynetoxin isolated from parasitized annual ryegrass on albumin and transferrin synthesis and secretion by cultured fetal rat hepatocytes

A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [³H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [³H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [³H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [³H]leucine but not [³H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM).     

STUDIES ON AMINO ACID LEVELS AND PROTEIN METABOLISM IN THE BRAINS OF GALACTOSE-INTOXICATED CHICKS1

Abstract— Levels of free amino acids, profiles of polyribosomes, and rates of protein synthesis and degradation were examined in the brains of chicks fed toxic levels of galactose. The content of a number of amino acids were altered; alanine and leucine were most strikingly depressed, whereas levels of aspartate were elevated. Polyribosomal profiles were unaltered. There appeared to be no detrimental effect on protein synthesis as judged by in vivo incorporation of L-[U-¹⁴C]leucine and L-[guanidino-¹⁴C]arginine. Likewise, the half-lives of proteins, measured by the loss of L-[guanidino-¹⁴C]arginine, were similar in experimental and control groups. In contrast, initial rates of incorporation of [³H]glucosamine into glycoproteins were enhanced. The effect was greatest in the microsomal fraction and typically 50 per cent greater than controls. Levels of free glucosamine and protein-bound hexosamine were essentially unaltered in the galactose-fed chicks.     

The sequential synthesis of the polypeptide chain of serum albumin by the microsome fraction of rat liver

1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [³⁵S]methionine, [¹⁴C]isoleucine or [¹⁴C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both ³H and ¹⁴C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [¹⁴C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Role of liver-cell potassium ions in secretion of serum albumin and lipoproteins

1. The incorporation of l-[1-¹⁴C]leucine into the proteins of liver slices and into the serum albumin and lipoproteins transported by these slices was investigated. 2. Transport rates were found to be dependent on the K⁺ content of the slices. 3. The effect of K⁺ on transport of serum albumin and of serum lipoprotein can be separated from any effect on synthesis by altering K⁺ concentrations after inhibition of protein synthesis by cycloheximide or puromycin. 4. The effect of low K⁺ concentrations is reversible. 5. There is linear relationship between the K⁺ content of the slices and the transport of protein. A simple method is described for maintaining various steady concentrations of K⁺ in the liver slices. 6. K⁺ may be replaced by Rb⁺. Cs⁺ is partly effective, but NH₄⁺ and Li⁺ are no more effective than Na⁺. 7. We found evidence that K⁺ content rather than the flux rates of K⁺ or Na⁺ is important in this effect. 8. These results are probably important in ethionine and carbon tetrachloride poisoning in the rat, and may be significant in liver transplantation.     

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY : II. Electrophoretic and Immunological Characterization of Microsomal Subfractions

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [³H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [³H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [³H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

Inhibition of protein synthesis and ATPase in mitochondria by the administration of the convulsant 3-mercaptopropionic acid.

¹⁴C leucine incorporation into proteins by cerebral cortex subcellular fractions was studied after the administration of the convulsant 3-mercaptopropionic acid (MP). It was found that MP decreased protein synthesis by isolated nerve endings and mitochondria but not by microsomal fractions. It was also observed that mitochondrial ATPase was inhibited. These findings suggest that the inhibition of protein synthesis might be an indication of a disequilibrium of the normal energy-yielding metabolism.     

LEUCINE OXIDATION IN BRAIN SLICES AND NERVE ENDINGS1

—It is generally believed that leucine serves primarily as a precursor for protein synthesis in the central nervous system. However, leucine is also oxidized to CO₂ in brain. The present investigation compares leucine oxidation and incorporation into protein in brain slices and synaptosomes. In brain slices from adult rats, these processes were linear for 90min and ¹⁴CO₂ production from 0·1 mm -l -[l-¹⁴C]leucine was 23 times more rapid than incorporation into protein. The rate of oxidation increased further with greater leucine concentrations. Experiments with l -[U-¹⁴C]leucine suggested that all of the carbons from leucine were oxidized to CO₂ with very little incorporation into lipid. Oxidation of leucine also occurred in synaptosomes. In slices, leucine oxidation and incorporation into protein were inhibited by removal of glucose or Na⁺, or addition of ouabain. In synaptosomes, replacement of Na⁺ by choline also reduced leucine oxidation; and this effect did not appear to be due to inhibition of leucine transport. The rate of leucine oxidation did not change in brain slices prepared from fasted animals. Fasting, however, reduced the incorporation of leucine into protein in brain slices prepared from young but not from adult rats. These findings indicate that oxidation is the major metabolic fate of leucine in brain of fed and fasted animals.     

Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1. The inhibition of incorporation of ¹⁴C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [¹⁴C]leucine and [¹⁴C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN : Localization of Newly Formed Proteins within the Endoplasmic Reticulum

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the ¹⁴C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Further studies of the transport of protein to nerve endings

Mice were injected intracerebrally with [l-¹⁴C]leucine, and the specific activities of subcellular fractions of brain and effractions of isolated nerve endings were determined. There was a progressive increase in the specific activity of protein associated with isolated nerve endings after incorporation of [l-¹⁴C]leucine into whole brain protein had terminated. Although, the incorporation of [¹⁴C]leucine into soluble protein of whole brain did not differ significantly in mice which were 3 months or 1-year old, the subsequent increase in specific activity of soluble protein isolated from nerve endings was significantly greater in the younger animals; 6-month-old mice were intermediate. Therefore, changes in some aspect of the transport of protein to nerve endings is altered even after sexual maturity. Anaesthetization with pentobarbitone during incorporation of [¹⁴C]leucine into protein, and inhibition of protein synthesis with acetoxycycloheximide after incorporation of [¹⁴C]leucine was complete, did not interfere with the subsequent appearance of radioactive protein at the nerve ending. Evidence is presented for the transport, from a proximal site of synthesis, of protein associated with particulate components of the nerve ending, including synaptic vesicles.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.     

Characterisation of amino acid incorporation by subcellular fractions from sterile beet disks

Summary The optimum concentrations of leucine, ATP, GTP and Mg²⁺ ion for the incorporation of leucine into protein by the microsomal fraction isolated from sterile disks of red beetroot are 0.06 mM, 5 mM, 0.5 mM, and 12 mM respectively. Incorporated ¹⁴C-leucine does not exchange with an excess of soluble-¹²C-leucine. Incorporation into protein is partly dependent on the addition of a high speed supernatant fraction which incorporates leucine into a product with the properties of aminoacyl RNA. Addition of polyuridylic acid to microsomes isolated from fresh disks stimulates the incorporation of phenylalanine into protein nine-fold but has no effect on leucine incorporation. Polyuridylic acid — stimulated incorporation is not inhibited by chloramphenicol. Preincubation of fresh microsomes with trypsin does not increase their activity. These results suggest that the low activity of fresh microsomes may be due to a lack of messenger RNA. The mitochondrial fraction shows a rise and fall in leucine-incorporating ability during aging similar to that shown by the microsomal fraction. Studies with inhibitors suggest that about 25% of this incorporation is due to the mitochondria themselves, the rest being attributable to large microsomes. Fractions isolated from disks aged under non-sterile conditions show large incorporations of leucine which are not dependent on an added energy source. This result confirms the importance of using aseptic techniques when studying the aging of storage tissue disks.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.