What Is the Signal for the Posttranslational Arginylation of Proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [³H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.     

N-Terminal Arginylation of Sciatic Nerve and Brain Proteins Following Injury

N-terminal protein arginylation has been demonstrated in vitro and in situ and has been reported to increase following injury to sciatic nerves of rats. The present study attempts to demonstrate these reactions in vivo by applying [³H]Arg to the cut end of sciatic nerves in anesthetized rats and assaying for N-terminal arginylation using Edman chemistry and acid precipitation of labeled proteins in the proximal nerve segment. No evidence was found for arginylation in an aqueous soluble fraction. However, N-terminal arginylation was detected in a urea soluble fraction at 2 hours after nerve crush. The data show that arginylation of rat sciatic nerve proteins occurs in vivo and suggest that the arginylated proteins formed an aqueous insoluble/urea soluble aggregate after arginylation. In other experiments, rat brains were injured and assayed for arginylation in vitro to test the hypothesis that injury causes an up-regulation of these reactions. Results showed an activation of the reaction at 2 hours post crush and indicate that increases in N-terminal arginylation are likely to be a general response to injury in nervous tissue.     

N-Terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [³H]Arg. Incorporation of [³H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [³H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [³H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro). However, in intact cells, doses as low as 10 uM Cx completely inhibited the incorporation of [³H]Arg/Lys into proteins. One uM Cx allowed for some incorporation of [³H]Arg/Lys into protein and approximately 40% of the Cx insensitive Arg was incorporated into the N-terminal. These results show that in vivo but not in vitro, Cx can block protein modification, suggesting that either in intact cells protein modification requires protein synthesis, or that Cx has effects other than as an inhibitor of protein synthesis on cells in culture, effects that it does not have on the partially purified components of the reaction.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Post-Translational Arginylation of Proteins in Cultured Cells

The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [¹⁴C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.     

Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat,Higher Metabolic Rate,Damaged Spermatogenesis,and Neurological Perturbations

In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway''s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ∼15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1^−/− spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.     

Major proteins secreted by the epididymis of Lacerta vivipara. Identification by electrophoresis of soluble proteins

During their period of activity, epithelial cells of the lizard epididymis produce secretory granules containing highly insoluble central cores of protein nature (protein H). After centrifugation of the epididymal fluid at 15 000 × g, major soluble proteins were separated in the supernatant by polyacrylamide gel electrophoresis. These proteins were labelled by repeated injections of [³H]leucine into animals. In cylindrical gel electrophoreses, labelled proteins migrated as a single band towards the anode in the presence of SDS, and as two separate bands without SDS. The fastest component obtained in non-denaturing conditions was designated protein L. In two-dimensional gel electrophoreses, the two bands separated in the first dimension both migrated to the same position in the second dimension with SDS. Consequently it may be assumed that protein L is a monomer (molecular weight about 16 000–20 000) able to aggregate into polymers which can be dissociated with SDS. It was proved by hemicastration experiments that these soluble proteins did not originate from the testis. In addition, they were detected after short incubation of epididymal tissues in the presence of [³H]leucine. It is concluded that these proteins are elaborated by epithelial cells of the epididymis and discharged into the lumen. A possible role in the physiology of spermatozoa is briefiy discussed.     

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. d-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only d-glucose and d-mannose were able to stimulate insulin release and cyclic [³H]AMP accumulation in the absence of other substrate. d-fructose had a stimulatory effect in the presence of 3.3 mM d-glucose only at a high concentration (38.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. d-Galactose was effective only together with 8.3 mM d-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [³H]AMP accumulation, was glucose-mannose-fructose-galactose.l-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM d-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [³H]AMP response.d-mannoheptulose and d-glucosamine inhibited the insulin and cyclic [³H]-AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s.Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [³H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the β-cell.     

Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Biosynthesis and processing of delta sleep-inducing peptide-like precursors in primary cultures of mouse anterior pituitary cells

The biosynthesis and processing of material resembling delta sleep-inducing peptide (DSIP) have been studied in mouse anterior pituitary primary cell cultures. Cells were pulse/chase incubated with 3H-labelled amino acids (Gly, Arg or Ala) and cell extracts were immunoprecipitated with DSIP antiserum. Labelled DSIP-related proteins were resolved by SDS/PAGE. Multiple forms of DSIP-immunoprecipitable material were observed, including three precursors of molecular mass 50-60 kDa which were processed to two major groups of intermediates of 35-45 kDa and 9-16.5 kDa. These intermediates appear to be processed to a DSIP-related peptide (molecular mass less than 3 kDa), which co-ran on reversed-phase HPLC with an endogenous form of DSIP in mouse anterior pituitary, but not with rabbit DSIP. This less than 3-kDa peptide incorporated [3H]Gly, but not [3H]Arg or [3H]Ala. In addition, it incorporated [3H]glucosamine, indicating that it was a glycopeptide. Secretion studies showed release of the less than 3-kDa DSIP-like glycopeptide and the 9-16.5-kDa group of intermediates into the medium. The present study demonstrates the biosynthesis of a small DSIP-like glycopeptide in mouse anterior pituitary cells, which is not identical with, but has similarities to, rabbit DSIP.     

Selective loss of a plasma membrane protein associated with contraction of skeletal muscle

Muscle proteins were labeled by incubating isolated frog sartorius muscles with [³H]- or [¹⁴C]phenylalanine. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions revealed a major protein band with an apparent molecular weight of approx. 96 000. Radioactivity in this band showed a clearly delineated decrease, relative to other bands, when previously labeled muscles were induced to contract either by electrical stimulation or by increasing the influx of Ca²⁺ from the incubation medium. It is postulated that a Ca²⁺-activated neutral protease may account for this decrease in labeled membrane protein.     

Palmitoylation of brain capillary proteins

Palmitoylation is a reversible posttranslational modification which is involved in the regulation of several membrane proteins such as β₂-adrenergic receptor, p21^ras and trimeric G-protein α-subunits. This covalent modification could be involved in the regulation of the numerous membrane proteins present in the blood-brain barrier capillaries. The palmitoylation activity present in brain capillaries was characterized using [³H]palmitate labeling followed by chloroform methanol precipitation. Palmitate solubilizing agents such as detergents and bovine serum albumin (BSA), were used for optimizing activity. Some palmitoylated substrates were identified using [³H]palmitate labeling followed by immunoprecipitation with specific antibodies. Two optimal palmitate solubilization conditions were found, one involves cell permeabilization (Triton X-100) and the other represents a more physiological condition where membrane integrity is conserved (BSA). Sensitivity to the cysteine modifier N-ethylmaleimide and to hydrolysis, using hydroxylamine or alkaline methanolysis, indicated that palmitic acid was bound to the proteins by a thioester bond. Maximal palmitate incorporation was reached after 30 or 60 min of incubation in the presence of Triton or BSA, respectively. Depalmitoylation was observed in the presence of BSA, but not with detergents. The palmitoylation reaction was optimal at pH 8 or 9 in the presence of Triton or BSA, respectively, but palmitoylated substrates were detectable over a wide range of pH values. In the presence of Triton X-100, the addition of ATP, CoA and Mg²⁺ to the incubation medium increased palmitoylation by up to 80-fold. Two palmitoylated substrates were identified, a 42 kDa G-protein α subunit and p21^ras. The study shows that the utilization of palmitate solubilizing agents is essential to measure in vitro palmitoylation in brain capillaries. Several palmitoylated proteins are present in the blood-brain barrier including five major substrates of 12, 21, 35, 42 and 55 kDa. It is suggested that palmitoylation could play a crucial role in the regulation of brain capillary function, since the two substrates identified in this study are known to be involved in signal transduction, vesicular transport and cell differentiation.     

Glutamine B16 insulin: Reduced insulin-like metabolic activity with moderately preserved mitogenic activity

An analogue of insulin in which the naturally occurring tyrosine residue in position B¹⁶ is replaced by a glutamine residue has been synthesized. Glutamine appears in the corresponding position in the B-domain of the insulin-like growth factors. This analogue displays 9% of the potency of insulin in binding to the insulin receptor from rat liver plasma membranes, 17% in stimulating the conversion of [3-³H] glucose into lipids in rat adipocytes, and 23% in insulin radioimmunoassay, but 40% of the potency of insulin in stimulating DNA synthesis in cultured chick fibroblasts. The analogue is a more potent mitogen than is a hybrid molecule which contains the A-chain of insulin and the entire B-domain sequence of IGF-I.

     

Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [³H]AMP ([³H]cAMP) and washed, and the bound [³H]cAMP was estimated. The band of [³H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [³H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [³H]-cAMP was bound in Peak II than in Peak I. The [³H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity.Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [³H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [³H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described.Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.     

Glutamine B16 insulin: Reduced insulin-like metabolic activity with moderately preserved mitogenic activity

An analogue of insulin in which the naturally occurring tyrosine residue in position B¹⁶ is replaced by a glutamine residue has been synthesized. Glutamine appears in the corresponding position in the B-domain of the insulin-like growth factors. This analogue displays 9% of the potency of insulin in binding to the insulin receptor from rat liver plasma membranes, 17% in stimulating the conversion of [3-³H] glucose into lipids in rat adipocytes, and 23% in insulin radioimmunoassay, but 40% of the potency of insulin in stimulating DNA synthesis in cultured chick fibroblasts. The analogue is a more potent mitogen than is a hybrid molecule which contains the A-chain of insulin and the entire B-domain sequence of IGF-I.     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.     

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD₅₀ of 1.1×10^–8M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [¹²⁵I]-insulin with an ED₅₀ of 1.1×10^–8 M, which is identical to the ED₅₀ of insulin (1.1×10^–8M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [¹²⁵I]-IGF-1 with a DTaI concentration of 1.1×10^–8 M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0±2.0 fmoles/mg protein down to 3.6±0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2±0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [¹²⁵I]-insulin and no apparent difference in [¹²⁵I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum