The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

Incorporation of labeled formate in rat liver mitochondrial proteins and initiation of protein synthesis

During incubation of rat liver mitochondria in vitro labeled formate is incorporated into TCA-insoluble substance during mitochondrial translation. Data from hydrolysis with CNBr (after methionine residues) or with 0.5 N HCl (deformylation of amino acid N-formyl derivatives) suggest that about half of the total protein radioactivity is incorporated in formate groups of N-terminal methionine. Labeling of growing polypeptides with formate (but not with phenylalanine or methionine) oscillates with a period of about 13 min. The potential initiation capacity is unchangeable and exceeds that observed experimentally by one order of magnitude. The data obtained are consistent with the previously proposed hypothesis no synchronization of mitochondrial protein synthesis which cannot be induced by the steps preceding the formation of the first peptide bound.     

Polyamine-induced DNA Synthesis and Mitosis in Oat Leaf Protoplasts

Freshly isolated protoplasts from leaves of oat seedlings (var. Victory) which do not divide when cultured on a wide range of media are capable of incorporating tritiated leucine, uridine, and thymidine into trichloroacetic acid-insoluble macromolecules. Over 70% of the leucine and uridine incorporated over an 18-hour period are found in protein and RNA, respectively, as shown by hydrolysis of the macromolecular products with a specific protease or RNase. In contrast, little or none of the tritiated thymidine is incorporated into macromolecules hydrolyzable by DNase over an 18- to 96-hour period. Incorporation of thymidine into trichloroacetic acid-insoluble material declines sharply with increasing time of culture after 18 hours. However, addition of diamines or polyamines to the medium not only prevents the decline, but actually increases net thymidine incorporation, including a fraction going into DNA. A significant increase in mitoses and binucleate protoplasts is also observed in 72- to 168-hour cultures.     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Tubulin does not bind glycerol irreversibly

Tubulin incorporates radioactivity when incubated with preparations of [¹⁴C]-glycerol, but the material that binds to tubulin is a contaminant rather than glycerol itself. The apparent binding of glycerol by tubulin was not stoichiometric, nor was it significantly affected by dilution of the radioactive glycerol preparation with unlabelled glycerol. Radioactive glycerol purified by prior exposure to protein was an order of magnitude less effective in binding to tubulin than unpurified glycerol. Cytochrome-C and bovine plasma albumin, as well as tubulin, incorporated radioactivity from isotopically labelled glycerol. The amount of radioactivity incorporated into tubulin was unaffected by assembly-disassembly.     

Carboxymethylation of a minor ribonuclease from Aspergillus saitoi.

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.     

Association of heparan sulfate proteoglycan and laminin with the cytoskeleton in rat liver

Rats were injected with 35SO4 and after 2 h their livers were removed and used to prepare a detergent-insoluble cytoskeleton fraction. Spectrin, cytokeratins, and actin were major protein components of the isolated cytoskeletons. The cytoskeleton fraction accounted for approximately 14% of the total trichloroacetic acid-insoluble 35SO4 radioactivity incorporated into the liver. The cytoskeleton-associated radioactivity was present in a single species of macromolecule. This molecule was not present to a significant extent in the detergent-soluble fraction containing the cell supernatant and dissolved membrane proteins. Further characterization revealed the cytoskeleton-associated molecule was a heparan sulfate proteoglycan: it was eluted from a Sepharose CL-4B column under denaturing conditions at Kav = 0.4; following mild alkaline hydrolysis the radioactivity was eluted at a Kav = 0.7; when this material was subjected to nitrous acid hydrolysis all of the radioactivity was eluted near the column included volume. The isolated cytoskeletons contained attached nuclei. Pure nuclei isolated without associated cytoskeletal elements contained less than 1% of the total liver trichloroacetic acid-insoluble 35SO4 radioactivity and no detectable heparan sulfate proteoglycan. These results suggested that other matrix proteins might be associated with the liver cytoskeleton. When the subcellular distribution of laminin was monitored by immunostaining proteins transferred to nitrocellulose, laminin was detected exclusively in the cytoskeleton fraction. These results provide evidence for an association between extracellular connective tissue proteins and intracellular structural proteins.     

Characterization of an alpha-glucan from chick embryo sternum whose synthesis is stimulated by L-3,5,3'-triiodothyronine and human serum.

Incorporation of [³H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l-3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 nm triiodothyronine and 5–20% ($\text{v}\text{v}$) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [³H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [³²P]phosphate, [³⁵S]sulfate, [³H]uridine, or [³H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.     

In vivo incorporation of [2-3H]-myo-inositol into frog opsin

The in vivo incorporation of [2-3H]-myo-inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U-14C]-myo-inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2-3H]-myo-inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.     

The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.     

The covalent attachment of polyamines to proteins in plant mitochondria.

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.     

Biosynthesis of a glycosylated keratin by human keratinocytes

Human keratinocytes, cultured in the presence of D-[1-14C]glucosamine, incorporated radioactivity into a cytoskeleton-associated glycoprotein with Mr 53,000. This glycoprotein co-purified with prekeratin when keratinocyte cytoskeletons were extracted with 0.1 M citric acid/0.1 M sodium citrate and subjected to isoelectric precipitation at pH 4.0. Analysis of the prekeratin polypeptides by two-dimensional gel electrophoresis revealed that the radioactivity was restricted to a single polypeptide with an isoelectric point in the pH range 4.5-5.5. Acid hydrolysis of prekeratin followed by paper chromatography of the hydrolysate showed that the radioactivity was incorporated as glucosamine and not by metabolic conversion to amino acids. Control experiments showed that the radioactivity associated with the glycoprotein of Mr 53,000 was not the result of adsorbed glycolipids or non-enzymatic labelling. In contrast to the incorporation of D-[1-14C]glucosamine and D-[6-3H]glucosamine, no appreciable amounts of L-[6-3H]fucose, D-[2-3H]mannose or 32PO4 were incorporated into this glycoprotein. The immunological relationship of the glycoprotein of Mr 53,000 to the keratins was demonstrated by its reactivity with both polyclonal and monoclonal antisera to keratin.     

Simulated hypogravity and proline incorporation into salt-extractable macromolecules from cell walls

Proline [U-¹⁴C] was fed to shoots of intact Tagetes patula grown normally, on horizontal clinostats, or on vertical clinostats rotating at 15 rev/hr. After various periods the incorporation of ¹⁴C into salt-extractable material from the cell walls of stems, petioles, leaves and flowers was determined. The cell walls of the gravity-compensated plants (grown on horizontal clinostats) has the highest amount of salt-extractable radioactivity. A 2- to 9-fold increase was observed in comparison to either the normal or vertical clinostat plant controls. Some physico-chemical properties of the salt-extractable fraction suggest that it consists of highly charged, low MW entities, possibly short chain peptides. On acid hydrolysis this material yields radioactive aspartic acid, glutamic acid and proline. The presence of labelled hydroxyproline is suggested. After acid hydrolysis of the cell walls of leaves, it was found that ca 4 times the amount of ¹⁴C was incorporated in the hypogravity-grown plant compared to the controls. It appears likely that extensibility changes in tissues under simulated hypogravity required additional cell wall protein.     

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.     

Biosynthesis ofSaccharomyces cerevisiae glycoproteins: Nature of some participating glycolipids

A particulate membrane preparation fromSaccharomyces cerevisiae catalyzed the incorporation of mannose from GDP-mannose into lipids that were extractable in chloroform-methanol. One lipid has been previously characterized as dolichyl phosphomannose. Another one was purified by chromatography on silicic acid, DEAE-cellulose and Sephadex LH-20 and found to be alkali unstable. The lipid moiety was shown to be dolichol and the glycosydic part contained mannose, glucose and glucosamine.Radioactive mannose was also incorporated at a slower rate into more polar compounds. They were soluble in chloroform-methanol-water and were seen to liberate neutral oligosaccharides after alkaline hydrolysis.Radioactive mannose was also incorporated into substances which behave chemically as glycoproteins since they were insoluble in organic solvents, water and trichloroactic acid. Pronase treatment of the trichloroacetic acidinsoluble material released water-soluble oligosaccharides.When the particulate preparation which had been extracted with chloroform-methanol at –20 C, was incubated with GDP-(U-¹⁴C)mannose, radioactivity was incorporated into glycolipids that were soluble in chloroform-methanol-water and into glycoproteins. This result suggests that at least part of the mannose was transferred to endogenous acceptors independent of dolichyl phosphomannose.     

Acetylation of Synaptosomal Protein: Inhibition by Veratridine

Abstract: Incubation of synaptosomes with [³H]acetate results in rapid labeling of protein. Labeling is decreased in the presence of veratridine, and the effect of veratridine is blocked by tetrodotoxin. Most of the radioactivity can be removed by base or acid hydrolysis, and is probably incorporated as acetate; it is this fraction that is affected by the veratridine. The data suggest that veratridine stimulates deacetylation of synaptosomal protein. This raises the question whether acetylation-deacetylation is involved in membrane function.     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

Acylation of bovine rhodopsin by [3H]palmitic acid

Bovine retinas or preparations of rod outer segments incorporate [3H]palmitic acid into rhodopsin. The incorporation is both time- and temperature-dependent. The major product retains the chromatographic and electrophoretic properties of rhodopsin and remains photosensitive as demonstrated by alteration of its chromatographic behavior upon exposure to light. The incorporated radioactivity resists extraction with organic solvents and is not dissociated from the protein by detergents or under the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive free fatty acid can, however, be released by alkaline hydrolysis. Hydroxylamine treatment yields a mixture of the free fatty acid and the fatty acyl hydroxamate. These results demonstrate the formation of an ester bond between [3H]palmitic acid and rhodopsin. Cycloheximide fails to inhibit the incorporation. This finding along with the ability of rod outer segments to support the incorporation point to the acylation of rhodopsin as a late post-translational event.     

Glycoprotein Synthesis in Plants: III. Interaction between UDP-N-Acetylglucosamine and GDP-Mannose as Substrates

This report presents evidence that enzymes present in crude extracts prepared from developing cotyledons of Phaseolus vulgaris can catalyze the transfer of radioactivity from UDP-N-[¹⁴C]acetylglucosamine into a chitobiosyl-lipid, lipid-oligosaccharide, and glycoprotein. Kinetic evidence supports the concept that the N-acetylglucosamine-containing lipids are precursors to the glycoprotein. Evidence is also presented which shows an interaction between GDP-mannose and UDP-N-acetylglucosamine when used as substrates for the synthesis of lipid-oligosaccharide and glycoprotein. Kinetic evidence, as well as isolation and characterization of the oligosaccharides released from lipid by mild acid hydrolyses, support the conclusion that mannose and N-acetylglucosamine are contained in the same oligosaccharide and that N-acetylglucosamine is present at the reducing end of the oligosaccharide. Ninety-eight per cent of the radioactivity which is incorporated from UDP-N-[¹⁴C]acetylglucosamine into the insoluble residue is solubilized by protease treatment. The glycopeptide released is quite similar in size and composition to the glycopeptide released by proteolytic digestion of vicilin, the major storage protein of Phaseolus vulgaris.