Rat alpha 1-microglobulin. Purification from urine and synthesis by hepatocyte monolayers

Rat alpha 1-microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin-A-Sepharose and ion-exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown-coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig alpha 1-microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross-reaction with human and guinea pig alpha 1-microglobulin was demonstrated. The concentration of alpha 1-microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n = 13) and rat serum alpha 1-microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric alpha 1-microglobulin and IgA. The latter alpha 1-microglobulin activity could be absorbed by anti-IgA serum. Rat alpha 1-microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and alpha 1-microglobulin was isolated from the medium by the use of immunoprecipitation with anti-(alpha 1-microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of alpha 1-microglobulin by the hepatocytes. The size of hepatic alpha 1-microglobulin was similar to that of purified urinary rat alpha 1-microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

In search of alpha 1-microglobulin on the lymphocyte surface.

alpha 1-Microglobulin was found by immunofluorescence not to be associated with human lymphoid and nonlymphoid cell lines. No accumulation of alpha 1-microglobulin was detected in culture media of these cell lines. A weak membrane fluorescence with anti-alpha 1-microglobulin on peripheral lymphocytes could not be blocked by the purified protein. No release of alpha 1-microglobulin into the growth medium was seen by normal cultured leukocytes. Treatment of normal lymphocytes, erythrocytes, and various cell lines with solubilization techniques did not yield any alpha 1-microglobulin. alpha 1-Microglobulin and protein HC display immunologic and biochemical identity. However, anti-protein HC stained almost all of the tested cell lines and normal lymphocytes. Blocking experiments with the purified protein were not successful. Antibodies reacting with a minor impurity (50,000 d) in the alpha 1-microglobulin or protein HC preparations could be absorbed from anti-alpha 1-microglobulin with normal leukocytes and a lymphoid cell line.     

Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum

1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an alpha-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-(14)C]Leucine or -valine and d-[1-(14)C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea-pig serum or fraction I, immunological identity being apparent with some lines. The microsome-bound substances thus represent serum glycoproteins or precursors of them. 8. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [(14)C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.     

Structural relationship between alpha 1-microglobulin from man, guinea-pig, rat and rabbit

Rabbit alpha 1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Sephadex G-100, affinity chromatography on concanavalin-A--Sepharose and ion-exchange chromatography on DEAE-Sephadex. Rabbit alpha 1-microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. Alpha 1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of alpha 1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human alpha 1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit alpha 1-microglobulin, with a gap between each band of 2.6--2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig alpha 1-microglobulin. Our results indicate that human alpha 1-microglobulin is substituted with two N-linked oligosaccharides, while only one is attached to each of the other alpha 1-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N-linked oligosaccharides carry the immunosuppressive activity of alpha 1-microglobulin.     

Neutral microprotein, a novel human plasma and urinary protein associated with a yellow-brown chromophore. Isolation from protein HC preparations and partial characterization

An apparently novel human plasma and urinary protein of low molecular weight was isolated from several highly purified preparations of protein HC by gel chromatography and high voltage electrophoresis with a yield of about 8 mg/g. The protein has a molecular weight of about 20,000, neutral electrophoretic mobility at pH 6.5 and a high content of half-cystine. It is associated with a yellow-brown chromophore like protein HC and could be demonstrated in all investigated preparations of isolated human, rabbit and guinea-pig protein HC and alpha 1-microglobulin.     

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.     

Isolation of plaice (Pleuronectes platessa) alpha1-microglobulin: conservation of structure and chromophore

A cDNA coding for plaice (Pleuronectes platessa) alpha1-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha1-microglobulin intracellularly. Recombinant plaice alpha1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of alpha1-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha1-microglobulin were similar to mammalian alpha1-microglobulin, indicating a high degree of structural conservation between fish and human alpha1-microglobulin. The monoclonal antibodies against plaice alpha1-microglobulin cross-reacted with human alpha1-microglobulin, emphasizing the structural similarity.     

Damage to protein synthesis concurrent with lipid peroxidation in rat liver slices: effect of halogenated compounds, peroxides, and vitamin E1

Protein synthesis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of oxidants and protein synthesis inhibitors. Protein synthesis by rat liver slices was evaluated by [3H]leucine incorporation into the trichloroacetic acid (TCA)-insoluble material, and lipid peroxidation was evaluated by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. Protein synthesis inhibition by bromotrichloromethane (BrCCl3) or t-butyl hydroperoxide (t-BOOH) depended on the incubation time and oxidant concentration. [3H]Leucine incorporation was decreased to 20 and 47% of control values and TBARS were enhanced from the control value of 16.9 to 45.3 and 62.5 nmol/g of liver by incubation for 1 h with 1 mM BrCCl3 and t-BOOH, respectively. Following incubation, both protein synthesis damage and lipid peroxidation were decreased in control and oxidant-treated slices prepared from rats injected with 200 mg of DL-alpha-tocopherol/kg of body wt. Release of lactate dehydrogenase was not enhanced by oxidant treatment. Protein synthesis inhibitors reversibly decreased [3H]leucine incorporation, but the effect of oxidants on protein synthesis was irreversible. Cumene hydroperoxide and methyl ethyl ketone peroxide, but not hydrogen peroxide, damaged protein synthesis and induced lipid peroxidation. The ability of carbon tetrabromide, benzyl chloride, bromoform, bromobenzene, carbon tetrachloride, chloroform, dichloromethane, and bromochloromethane to inhibit protein synthesis was correlated with their ability to induce lipid peroxidation, and with their LD50. The results suggest that oxidant-induced lipid peroxidation and protein synthesis damage occurred concurrently, and that protein synthesis inhibition may be involved in cell injury or death mediated by free radicals.     

In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [³H]serine and [¹⁴C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell.     

Differential long-term effects of d-chloramphenicol on the biogenesis of mitochondria in normal and regenerating rat liver

1. Normal and partially hepatectomized rats (150g) were injected daily with d-chloramphenicol (20mg) for a period of 4 weeks, in order to investigate whether defective mitochondria could be induced in vivo in higher organisms as in yeast, and to measure the degree of inhibition of the mitochondrial function thus obtained. 2. The antibiotic did not affect growth and increased the amount of liver protein without changing the mitochondrial yield. 3. The respiration of isolated mitochondria from regenerated liver (regeneration completed) with succinate, α-oxo-glutarate, isocitrate and malate, was decreased in the chloramphenicol-treated rats, whereas in normal liver the antibiotic increased the mitochondrial oxygen consumption with succinate and did not significantly change the respiration with other substrates. 4. Mitochondrial cytochromes and respiratory enzymes were also decreased in amount in regenerated liver from the treated rats and enhanced in normal liver. 5. The protein specific radioactivities of most mitochondrial and microsomal subfractions, 30min after an injection of [¹⁴C]leucine, were decreased in regenerated liver under the action of chloramphenicol. Conversely, the incorporation of [¹⁴C]leucine into proteins of most subfractions in incubations of liver slices was enhanced in the case of normal rats treated with the antibiotic. 6. It is concluded that in regenerated liver chloramphenicol induces functionally defective mitochondria by inhibiting their biogenesis, whereas in normal liver the stimulation of respiration and protein synthesis is probably a secondary detoxication response.     

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.     

Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.     

Studies on the assembly and secretion of a multicomponent protein. The biosynthesis of vitellogenin, an oestrogen-induced glycolipophosphoprotein

1. Incorporation of [(32)P]P(i) and [(3)H]leucine into vitellogenin secreted in vitro by liver slices from oestrogen-treated Xenopus laevis is accompanied by a 2h lag; no lag is apparent for the incorporation into total tissue protein. 2. The addition of cycloheximide was found immediately to inhibit further incorporation of radioactive leucine into total tissue protein. The incorporation into secreted vitellogenin, however, continued for 2h after the addition of cycloheximide. 3. Pulse-labelling of liver slices with [(3)H]leucine for 30min, followed by a chase with a large excess of unlabelled leucine, resulted in the appearance of radioactivity in secreted vitellogenin from 90min after the end of the pulse period. 4. Evidence is presented which suggests that of the radioactivity from [(3)H]leucine incorporated into proteins by the liver of oestrogen-treated Xenopus some 70% is present in the single protein vitellogenin. 5. The incorporation of [(32)P]P(i) into vitellogenin followed a pattern identical with that found for [(3)H]leucine in the pulse-labelling experiments and this indicates that synthesis of the polypeptide chain and incorporation of P(i) are closely linked processes. 6. The cumulative evidence suggests that the 2h lag phase represents the time required for the assembly and secretion of this multicomponent protein.     

Stimulation of cholinephosphotransferase activity by phosphatidylcholine transfer protein. Regulation of membrane phospholipid synthesis by a cytosolic protein

The effect of rat liver phosphatidylcholine transfer protein on the incorporation of CDP-choline and dioleoylglycerol into phosphatidylcholine catalyzed by rat liver microsomal CDP-choline: 1,2-diacyl-sn-glycerol cholinephosphotransferase was studied. In the presence of phosphatidylcholine transfer protein, the incorporation of CDP-choline into phosphatidylcholine was markedly stimulated. Phosphatidylcholine transfer protein isolated from either rat or bovine liver was capable of this stimulatory effect; in contrast, phosphatidylinositol transfer protein from rat liver had no effect on phosphatidylcholine synthesis. Kinetic analysis showed that microsomal phosphatidylcholine synthesis increased 2.4-fold after 1 min and reached a maximum of approximately 10-fold within 10 min in the presence of phosphatidylcholine transfer protein; in the absence of this protein phosphatidylcholine synthesis stopped after 2-4 min. These results suggest that phosphatidylcholine transfer protein permits phosphatidylcholine synthesis to proceed further. With the addition of phospholipid vesicles, as an acceptor membrane in the reaction mixture, there was a significant amount of protein-mediated transfer of synthesized phosphatidylcholine to the vesicles. Measurable transfer of synthesized phosphatidylcholine to vesicles could only be detected after a lag of 2-4 min. The stimulation of cholinephosphotransferase could be nearly abolished by increasing the amount of added phospholipid vesicles; concurrently, a greater transfer to the vesicles was observed. These results describe a new property of phosphatidylcholine transfer protein which may be of physiological significance in the regulation of phosphatidylcholine synthesis in mammalian tissues.     

Protein synthesis in central nervous tissue: studies on retina in vitro

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.     

Studies on the effect of 1-deoxynojirimycin on the release of albumin, sialyltransferase and alpha 1 acid glycoprotein from liver slices from normal and inflamed rats

Liver slices from control and 24hr inflamed rats were incubated for up to 20hr with 5mM 1-deoxynojirimycin (DN), an inhibitor of the processing glucosidases. The amounts of albumin and alpha 1-acid glycoprotein (AGP) and the activities of sialyltransferase were determined in liver and medium. The presence of DN significantly inhibited the release of AGP and sialyltransferase. The inhibitory effect of DN was most pronounced with slices from inflamed rats. Secretion of albumin was not inhibited. Incorporation studies with labelled leucine and mannose showed that the inhibitor did not significantly affect protein synthesis, but it did inhibit mannose incorporation into AGP and sialyltransferase. The results show that DN inhibits the secretion of acute phase AGP and sialyltransferase in liver slices and further suggests that sialyltransferase is a glycoprotein.     

Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins

A method for the in vitro perfusion of isolated guinea-pig mammary tissue is described that allows the radiolabelling of secretory and membrane proteins. Glands were depleted of methionine, labelled with [35S]methionine for 5 min and perfused with medium containing an excess of unlabelled methionine for varying times. The structural integrity of the alveoli in the perfused glands appeared well maintained. Epithelial polarity was preserved and junctional complexes were evident. About 20% of the methionine provided in the medium was extracted by glands of 10 g wet weight under the labelling conditions employed. With chase periods from 15 to 40 min, 50-70% of the methionine was incorporated into trichloroacetic-acid (TCA)-precipitable material. The principal radiolabelled proteins recovered from the tissue fractions had Mrs and isoelectric points similar to the major secretory proteins (i.e. caseins and alpha-lactalbumin) of guinea-pig milk. Autoradiography of tissue sections at the resolution of the light microscope showed that secretory proteins were transported from sites of synthesis within secretory cells to the alveolar lumina after 45 min. These highly labelled secretory proteins could be almost completely removed from microsomal fractions by treatment with sodium carbonate solutions. Proteins with Mrs from 30 000 to 200 000 were detected in the washed membranes by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These labelled membrane-associated proteins persisted in the microsomal membrane fraction after chase periods from 7.5 to 40 min.     

Translational control of protein synthesis in stimulated WI-38 fibroblasts.

A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function.     

Studies on the effect of inflammation on the acute phase response using rat liver slices

Liver slices from control and inflamed rats were incubated in McCoy's medium and incorporation of [3H]leucine into liver and medium proteins and into albumin and alpha 1-acid glycoprotein was monitored over 48 hr. The release of the new acute phase reactant, sialyltransferase was also monitored in this system. Earlier observations in which liver slices were incubated for 6 hr showed that increased leucine incorporation into liver and medium proteins and alpha 1-acid glycoprotein, coupled with decreased incorporation into albumin, correlated with the acute phase response of these proteins. Increased incorporation of leucine into these proteins was found following 48 hr incubation in McCoy's medium showing that slices were able to express the changes characteristic of the acute phase response over this longer time period of incubation. Sialyltransferase was released into medium in a linear fashion up to 15 hr and continued to increase for 30 hr in this system; there was a substantial increase in release of enzyme activity from slices from inflamed rats when compared to controls. Monokine-conditioned medium prepared from peritoneal exudate cells isolated from rats at various times after lipopolysaccharide administration was used to induce the acute phase response by intraperitoneal injection. Slices were prepared from these rats and sialyltransferase release from slices was monitored. Monokines prepared from peritoneal exudate cells isolated from rats at about 30 hr were most effective in stimulating sialyltransferase release from liver slices.     

Role of protein degradation in the growth of livers after a nutritional shift.

Fractional rates of synthesis and degradation of liver porteins were estimated during the rapid restoration of liver mass observed in protein-depleted mice when they are fed with an adequate diet. 1. Net protein gain was fastest 12h after the nutritional shift, when it reached a rate of 48% per day. 2. The RNA/protein ratio in livers of protein-depleted animals was essentially the same as in normal livers; it increased by a maximum of 13% 12h after the nutritional shift. 3. Rates of protein synthesis in vivo were measured by the incorporation into liver protein of massive amounts of L-[1-14C]leucine. In protein-depleted animals, the rate of synthesis per mg of RNA was 72% of that in normal livers. Normal rates were recovered within 12h of the nutritional shift. 4. The fraction of newly synthesized protein retained by the liver was studied after they were pulse-labelled by the intravenous injection of radioactive leucine, and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis); 3h later the livers in both experimental situations retained 58% of the newly synthesized protein. 5. Fractional rates of protein degradation were estimated either from the difference between the synthesis of stable liver proteins and the net protein increase, or by the disappearance of radioactivity from the liver protein previously labelled by the administration to the mice of NaH14CO3. Both procedures demonstrated a large decrease in the rate of protein degradation during liver growth.