Some observations on protein metabolism in chromosomes of non-dividing cells

1. The metabolism of chromosomal proteins has been studied in the pancreas, liver, and kidney of adult mice (a) by measuring the rates of glycine-N¹⁵ incorporation into histones and residual chromosome fractions, and (b) by measuring the extent to which N¹⁵, once incorporated into chromosomal proteins, is retained. 2. The uptake of isotopic nitrogen by these nuclear constituents was compared with that of protein fractions prepared from the cytoplasm by differential centrifugation in sucrose solutions. One such fraction, which comprises the bulk of the ribosenucleoprotein of the cell sediments as a pellet on high speed centrifugation. The supernatant remaining after this centrifugation is a fraction which, in the pancreas, is rich in the secretory enzymes synthesized by the cell. 3. A comparison of the rates of glycine-N¹⁵ uptake shows that cytoplasmic ribonucleoprotein is the most active of the protein fractions analyzed. In the pancreas it meets the conditions required of a precursor for the secretory enzymes of the supernate. 4. In all tissues considered the rates of glycine-N¹⁵ uptake into histone and residual chromosome fractions are lower, that for histone being the lowest of any of the protein components considered and that for residual protein approximating the over-all rate for cytoplasmic protein. 5. The effects of feeding and fasting upon glycine-N¹⁵ incorporation have been studied. In the pancreas, feeding causes a sharp increase in N¹⁵ uptake by the mixed tissue proteins and by the nucleoprotein and supernatant protein of the cytoplasm. There is a parallel increase in N¹⁵ uptake by the chromosomal constituents—histone and residual protein. 6. A parallelism between N¹⁵ uptake in cytoplasmic and chromosomal proteins is also observed in the liver and kidney when over-all protein metabolism is altered by feeding and fasting. 7. The responsiveness of the histones and residual proteins to changes in the environment has also been demonstrated in N¹⁵ retention experiments. The loss of isotope once incorporated into chromosomal proteins is much more rapid in fed than in fasted animals.     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

Synthesis of protein in the pancreas. III. Uptake of glycine-N15 by the trypsinogen and chymotrypsinogen of mouse pancreas

The uptake of glycine-N¹⁵ into the trypsinogen and chymotrypsinogen of mouse pancreas is much higher than that into any ribonucleoprotein component of the pancreas that has so far been investigated.     

Inhibition of heme synthesis in bone marrow cells by succinylacetone: effect on globin synthesis

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.     

Uptake of glycine-N15 by components of cell nuclei

1. The uptake of glycine-N¹⁵ by components of cell nuclei was studied. The nuclear components were derived both from tissues with high metabolic rates-mammalian liver, kidney, and pancreas-and from cells with relatively low rates of metabolism-avian erythrocytes and echinoderm sperm. N¹⁵ uptake by nuclear components of liver, kidney, and pancreas was far more rapid than by those of erythrocytes and sperm. 2. The nuclear components of liver, kidney, and pancreas for which measurements were made were DNA, histone, and residual protein of chromatin. Uptake into DNA was low, into histone higher, and into residual protein much higher still, being comparable with that into mixed cytoplasmic protein. 3. A comparison of the uptake of N¹⁵ by the chromosomal components, histone and DNA of liver, pancreas, and kidney showed that chromosomal "activity" varies in different cells and also in the same cell depending upon its over-all activity.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

Erythrocytes anion transport and oxidative change in β‐thalassaemias

β‐Thalassaemia is characterized by a decrease in globin β‐chain synthesis and an excess in free α‐globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid–base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and β‐thalassaemia‐like cells and tested for sulfate uptake. To test the antioxidant effect of Mg²⁺, other experiments were performed using normal and pathological cells in the presence of Mg²⁺. The oxidant status in thalassaemic cells was verified by increased K⁺ efflux, by lower GSH levels and by increased G6PDH (glucose‐6‐phosphate dehydrogenase) activity. The rate constant of SO₄ ^2? uptake decreases in thalassaemic cells as well as in β‐thalassaemia‐like cells when compared with normal cells. It increases when both cells are incubated with Mg²⁺. Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg²⁺ could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Accumulation of heme in mitochondria from rabbit reticulocytes with inhibited globin synthesis

Incubation of rabbit reticulocytes with cycloheximide and ⁵⁹Fe bound to transferrin in plasma induces excessive non-hemoglobin ⁵⁹Fe-labeled heme accumulation in mitochondria. During incubation of these mitochondria in vitro a part of ⁵⁹Fe-labeled heme is released into the surrounding medium. The addition of globin or bovine serum albumin to the incubation mixture essentially increases the amount of heme released from mitochondria.     

Induction of globin mRNA accumulation by hemin in cultured erythroleukemic cells

The role of heme in erythroid development is investigated in erythroleukemic (Friend) cells. Exogenous hemin induces the accumulation of globin mRNA and globin protein in T3-Cl2 erythroleukemia cells to levels comparable to those induced by polar solvents, such as dimethylsulfoxide (DMSO). The hemin concentration required for maximal induction (10^?4 M) is the same as that which stimulates globin message translation in reticulocytes or cell-free reticulocyte lysates. Hemin and DMSO together cause T3-Cl2 cells to accumulate 8–9 fold more globin mRNA than either inducer individually. The kinetics of globin mRNA induction in hemin as compared to DMSO are very different: globin message accumulation begins 4 hr after hemin addition, but not until 30–40 hr after DMSO addition. Biliverdin induces 20–40 fold less hemoglobin than hemin; delta-aminolevulinic acid and porphobilinogen do not induce.     

Structure of Annelid High Molecular Weight Hemoglobins (Erythrocruorins)

The extracellular vascular hemoglobins (erythrocruorins) ofannelids are polymeric oxygen carriers with molecular weightsof approximately 3 x 10⁶ or about 46 times the molecular weightof a vertebrate hemoglobin tetramer. The molecule appears asa dodecamer of 12 large submultiples arranged at the verticesof two regular hexagons one on top of the other in electronmicrographs. The dimensions are about 250 Å across theface of the hexagon and about 170 Å in height. The molecularweight of a one-twelfth submultiple is approximately 250 000.Biochemical studies suggest that each submultiple contains 16to 18 subunits and that the intact hemoglobin molecule containsapproximately 200 subunits. Unlike vertebrate hemoglobin whichcontains one heme moiety for each polypeptide chain the annelidhemoglobins apparently contain one heme per 15 to 20 chains.The reasons for this lack of a 11 heme chain stoichiometry arenot known at present. One possibility may be that it is theresult of insufficient purification of the hemoglobin. Alternatively,more than one globin chain might share a heme certain globinchains might lack the heme moiety and have a non hemoglobinfunction, or certain globin chains may lose their heme duringpurification of the hemoglobin. We are presently determiningthe amino acid sequence of one globin chain of Lumbricus terrestrishemoglobin. This information should be helpful in understandingthe structure of these interesting polymers.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

The onset of hemoglobin synthesis in spleens of irradiated mice after bone marrow transplantation.

Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.