Rapid Protein Synthesis Research Articles

Accelerated chemical synthesis of peptides and small proteins.

The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the "difficult" C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize "difficult couplings," rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods.

Tumour necrosis factor-alpha stimulates increased expression of prostaglandin endoperoxide H synthase Type 2 mRNA in amnion-derived WISH cells.

We have evaluated the mechanism by which tumour necrosis factor-alpha (TNF-alpha) induces increased prostaglandin (PG) biosynthesis in amnion-derived WISH cells. WISH cells were treated with 50 ng/ml TNF-alpha or vehicle for 0-24 h. PGE2 production was stimulated by TNF-alpha within 2 h and continued to accumulate for at least 24 h. Increased prostaglandin endoperoxide H synthase (PGHS)-2 mRNA expression was evident within 30 min and was highest by 1 h, returning to unstimulated levels by 2 h. The PGHS-2 mRNA was re-induced at 8 h and was also elevated at 16 h. Immunoreactive PGHS-2 protein was nearly undetectable in control cells. However, within 30 min of TNF-alpha treatment, PGHS-2 protein was elevated and was induced for at least 16 h suggesting rapid production of both the PGHS-2 mRNA and protein. Transcription run-on assays indicated that the initial increase in the PGHS-2 mRNA was due to a 20-fold increase in the rate of transcription. The PGHS-2 mRNA decayed with an apparent half-life of 1 h in TNF-alpha-stimulated WISH cells. Induction of PGHS-2 expression proceeded in the presence of 10 microg/ml cycloheximide which agrees with the classification of PGHS-2 as an immediate early gene. These results indicate that a bi-phasic induction of the PGHS-2 mRNA is due, in part, to an initial transcriptional activation which results in rapid and continued synthesis of the PGHS-2 protein. This may be a unique characteristic of amnion cells which may be partially responsible for increased PG concentrations in the amniotic fluid during infection-associated preterm labour.

Induction of Tumor Necrosis Factor-α In Vivo by a Skin Irritant, Tributyltin, Through Activation of Transcription Factors: Its Pharmacological Modulation by Anti-inflammatory Drugs

Skin irritant reactions are under the control of a network of cytokines and lipid mediators. This study characterized the production of tumor necrosis factor-alpha (TNF) induced by a skin irritant treatment, tributyltin (TBT), in mice through transcription factor activation and its pharmacologic modulation by anti-inflammatory agents. The ears of BALB/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. At different times thereafter, TNF production was analyzed both at the mRNA and protein level, by semiquantitative RT-PCR and L929 cytotoxicity assay, respectively. TBT induced rapid (1 h) TNF gene expression and protein synthesis. Maximal TNF production was observed 2 h after treatment. The production of TNF was paralleled by accumulation of skin water; this was partially prevented by intraperitoneal injection of antibody against murine TNF. These data indicate that skin irritation induced by TBT is attributable, in addition to the actions of other inflammatory mediators, to the action of keratinocyte-derived TNF. TNF production was preceded by a rapid (5 min) activation of nuclear factor-kappaB (NF-kappaB), which was also maximal 30 min after treatment. TBT-induced accumulation of skin water and TNF production were significantly reduced by topical treatment with dexamethasone and pentamidine, two anti-inflammatory agents. Interestingly, dexamethasone, but not pentamidine, decreased TBT-induced NF-kappaB activation, confirming in vivo that the glucocorticoid receptor interacts functionally within the nucleus with other transcription factors opposing one another's activity.

Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Maintenance of cellular levels of G-proteins: different efficiencies of alpha s and alpha o synthesis in GH3 cells.

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein alpha, beta and gamma subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, alpha s and alpha o, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of alpha s protein than to produce the same amount of alpha o protein. Such a situation could arise from a relatively rapid rate of alpha s protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of alpha s mRNA compared with alpha o mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for alpha s than for alpha o, yet the rate of alpha s protein synthesis is only 9 times greater than alpha o protein synthesis. The degradation rates of the two proteins are similar (13 h for alpha s and 18 h for alpha o). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of alpha s than alpha o, we asked whether there is a pool of alpha s mRNA that does not participate in protein synthesis. We found that virtually all alpha s and alpha o mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of alpha s protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.

T-cell Activation Leads to Rapid Stimulation of Translation Initiation Factor eIF2B and Inactivation of Glycogen Synthase Kinase-3

Mitogenic stimulation of T-lymphocytes causes a rapid activation or protein synthesis, which reflects in part increased expression of many translation components. Their levels, however, rise more slowly than the rate of protein synthesis, indicating an enhancement of the efficiency of their utilization. Initiation factor eIF2B catalyzes a key regulatory step in the initiation of translation, and we have therefore studied its activity following T-cell activation. eIF2B activity rises quickly, increasing as early as 5 min after cell stimulation. This initial phase is followed by an additional slow but substantial increase in eIF2B activity. The level of eIF2B subunits did not change over the initial rapid phase but did increase at later time points. Northern analysis revealed that levels of eIF2B mRNA only rose during the later phase. The rapid activation of EIF2B following mitogenic stimulation of T-cells is therefore mediated by factors other than its own concentration. The largest (epsilon) subunit of eIF2B is a substrate for glycogen synthase kinase-3 (GSK-3), the activity of which rapidly decreases following T-cell activation. Since phosphorylation of eIF2B by GSK-3 appears to inhibit nucleotide exchange in vitro, this provides a potential mechanism by which eIF2B may be activated.

Ca2+-independent arachidonic acid release by vascular endothelium requires protein synthesis de novo

We investigated the mechanism by which the G-protein activators aluminium fluoride and vanadate stimulate arachidonic acid release in pig aortic endothelial cells. Our previous study demonstrated a novel Ca(2+)-independent pathway of phospholipase A2 (PLA2) activation stimulated by aluminium fluoride in this model. In the present study, we found that sodium metavanadate stimulated a rapid concentration-dependent release of [3H]arachidonic acid from prelabelled cells. A more than 3-fold enhancement of arachidonic acid release was achieved in cells treated with 1 mM vanadate for 20 min. Synthesis of prostaglandin products was similarly enhanced. The release of arachidonic acid was not dependent on the presence of extracellular Ca2+, but did require protein synthesis de novo. Both cycloheximide and actinomycin D completely blocked aluminium fluoride- and vanadate-stimulated arachidonic acid release. Because fluoride and vanadate are known protein tyrosine phosphatase inhibitors, it is possible that PLA2 activation occurred secondarily to changes in protein tyrosine phosphorylation. Both aluminium fluoride and vanadate stimulated the rapid phosphorylation of 58, 93 and 120 kDa tyrosine-containing protein substrates. However, in contrast with arachidonic acid release, this response was found to be sensitive to the presence of extracellular Ca2+ and insensitive to blockers of protein synthesis de novo. Furthermore H2O2 treatment resulted in rapid tyrosine phosphorylation of the same substrates without a concomitant increase in arachidonic acid release. These results suggest that the effects of aluminium fluoride and vanadate on PLA2 are not due to changes in protein tyrosine phosphorylation, but do require rapid protein synthesis de novo.

Synthesis of Preparative Amounts of Biologically Active Interleukin-6 Using a Continuous-Flow Cell-Free Translation System

Cell-free synthesis of preparative amounts of interleukin-6 (IL-6) in a wheat germ extract is described. Following transcription of uncapped mRNA by SP6 polymerase, protein was synthesized in a continuous flow of translation components. The system remained active for at least 50 h, producing 1.25 × 106 units of IL-6. Recovered IL-6 was >80% pure, reacted with anti-IL-6 polyclonal antiserum in Western blots, and had a specific activity comparable to recombinant IL-6 purified from Escherichia coli. This is the first time biologically active IL-6 has been prepared in preparative amounts using a continuous-flow cell-free translation system and confirms the feasibility of using enhanced in vitro translation for rapid synthesis of proteins from cloned templates.

Heat shock increases chilling tolerance of mung bean hypocotyl tissue

The effects of heat shock on the chilling tolerance of mung bean [Vigna radiata (L.) Wilczek] seedling tissue were studied by using two measurements of chilling injury: increased 1‐aminocyclopropane‐1‐carboxylic acid (ACC) oxidase activity and solute leakage. ACC oxidase activity (measured as ACC‐induced ethylene production) of freshly excised mung bean hypocotyl segments was highly dependent on the temperature at which the seedlings were grown. However, this highly temperature‐dependent level of ACC oxidase activity was probably a wound response since it was almost entirely eliminated by incubating the excised segments at 20°C for 3 h. In contrast, heating of excised segments to 40°C for up to 4 h resulted in a time‐dependent increase in ACC oxidase activity which was sensitive to cycloheximide, indicating rapid protein synthesis during the heat treatment. ACC oxidase activity fell sharply during subsequent chilling at 2. 5°C. After 3 days of chilling, all treated segments, regardless of their initial ACC oxidase activity, showed a decline to the same low activity level and ACC oxidase activity continued to fall slowly for up to 9 days at 2. 5°C. Hypocotyl segments excised from seedlings held at 15°C showed no change in solute leakage, but leakage increased rapidly when seedlings were either chilled at 2. 5°C or heated to 32°C (just below the heat shock temperature). Chill‐induced leakage from non‐heat‐shocked segments increased steadily with chilling duration and was unaffected by cycloheximide concentration up to day 6. Within the elevated rate of leakage on day 9, however, leakage was lower from segments exposed to 10 and 50 μM cycloheximide. Solute leakage was markedly reduced for up to 9 days when segments were heat shocked at 40°C for 3 or 4 h with or without 10 M cycloheximide, but the presence of 50 μM cycloheximide caused an initial doubling of solute leakage and a 3‐fold increase after 3 days of chilling. Cycloheximide prevented formation of heat shock protection against chilling from the start at 50 μM and after 9 days at 10 μM. These results indicate that the protection afforded by heat shock against chilling damage is quantitative and probably involves protein synthesis.

The Heat Shock Response in a Photoautotrophic Cell Culture of Chenopodium rubrum: The Effects of Temperature and Light

Summary Heat shock treatment (HS) of the photoautotrophic cell culture of Chenopodium rubrum leads to a rapid synthesis of specific heat shock proteins (HSPs These HSPs belong to the well-known classes of the HSP80-, HSP70- and HSP20-families, and are fairly stable proteins. The steady-state concentration of the specific HS mRNAs, as well as the amount of in vivo labelled HSPs, show similar increments with increasing temperature up to 38 °C at standard light intensities, indicating a regulation primarily at the level of transcription. The effect of light intensities on the induction of HSP was investigated. It was found that with increasing light intensities the temperature at which the maximal HSP induction is achieved declines from 40 °C to 36 °C. Since high light intensities alone cannot induce HS gene expression, but can stimulate the HS response, we propose that light acts as a co-effector on HS transcription and might help to prevent cell damage from the adverse effects of light.

Complement membrane attack complexes induce in human leukemic cells rapid expression of large proteins (L-CIP)

The effect of sublytic doses of the complement membrane attack complexes (MAC) 1 on protein synthesis in human leukemic cells was examined. As shown herein, rapid protein synthesis is evident in K562 erythroleukemic cells upon exposure to sublytic complement doses. Analysis of cell extracts by SDS-PAGE revealed high molecular weight proteins which appeared in the cells already after 15 min treatment with complement at 37°C, reaching a maximal level after 40–50 min. These large complement-induced proteins (L-CIP) were clearly observed in gels stained by Coomassie blue and in autoradiograms following [ 35S]-Met or [ 3H]-Leu incorporation. Rabbit antibodies prepared against L-CIP were reactive in immunoassays with extracts of MAC-treated cells but not of non treated cells. They also bound to the surface of intact K562 cells (as determined by immunofluorescence), but only after treatment of the cells with complement. Both heterologous (rabbit and guinea pig) and homologous (human) sera induced L-CIP synthesis. The induction of L-CIP was indeed mediated by the complement MAC since L-CIP could not be detected in K562 cells exposed to heat-inactivated human serum or C6-deficient rabbit serum. Similarly, C7- or C8-deficient human sera could not induce L-CIP production unless they were reconstituted with purified human C7 or C8, respectively. The synthesis of L-CIP was largely inhibited by the protein synthesis inhibitors cycloheximide and puromycin and partially inhibited by the RNA synthesis inhibitor actinomycin D. L-CIP was similarly induced in two other human leukemic cell lines, U937 and HL-60, but not in K562/S, a subline of K562 which is highly sensitive to complement damage. These results are discussed with respect to the resistance of leukemic cells, and nucleated cells in general, to complement-mediated immune damage.

Light and the recovery from heat shock induce the synthesis of 38 kDa mitochondrial proteins in Neurospora crassa.

The effect of light on the protein synthesis pattern in the mitochondria of Neurospora crassa was examined by in vivo labelling with [35S]-methionine and two-dimensional gel electrophoresis. A brief 5-min illumination induced the rapid and transient synthesis of a 38-kDa protein. White collar-mutants were not stimulated to synthesize this protein by light. A protein of a similar molecular weight and isoelectrical point was synthesized during recovery from heat shock.

Molecular cloning and characterization of a cDNA encoding a water stress protein (WSP23) from wheat roots

Plants respond to different environmental stress stimuli by rapid synthesis of stress proteins which are hypothesized to have a protective function in cellular metabolism. The nucleotide sequence of a wheat ( Triticum aestivum L.) cDNA encoding one such protein, WSP23 whose transcripts rapidly accumulate during water stress is presented. Sequence analysis of this cDNA yielded a derived polypeptide, 231 amino acids in length, with a predicted molecular mass of 23 229 and isoelectric point (pI) of 10.8. The putative polypeptide is highly hydrophilic and has highly conserved amino acid repeats that are similar to several other water stress responsive proteins found in higher plants. Genomic Southern blot hybridization data suggest the presence of a small multigene family encoding these proteins in wheat. Coordinate expression of genes that encode three different groups of water stress proteins indicates that the molecular changes that occur in plants in response to water deficits may mimic the gene expression pattern that occurs during late embryo maturation stages.

Acute-Phase Proteins during Hemodialysis: Correlations with Serum Interleukin-1β Levels and Different Dialysis Membranes

The effects of hemodialysis (HD) on the levels of serum amyloid A (SAA), C-reactive protein (CRP) and interleukin-1 beta (IL-1 beta) were studied in 8 patients. Bicarbonate dialysate was used exclusively, and three different membranes, Cuprophan (CU), cellulose acetate (CA), and polymethylmetachrylate (PMMA) were compared. The SAA levels increased significantly with each membrane. With CU, they rose from 4.0 +/- 2.0 (mg/l, mean +/- SEM) to 9.6 +/- 2.8 at 60 min and to 15.0 +/- 4.9 at 240 min. The values with CA were 3.8 +/- 2.1, 15.3 +/- 5.6, and 23.8 +/- 3.9; and with PMMA 2.4 +/- 1.3, 12.1 +/- 5.6, and 12.1 +/- 5.9, respectively. The alterations of SAA neither correlated with the weight loss nor the increase of serum albumin during dialysis. The CRP values showed insignificant changes. The IL-1 beta levels rose with CU from 87 +/-18 (ng/l) to 155 +/- 33 at 60 min and to 172 +/- 47 at 240 min. With CA, the values were 67 +/- 14, 198 +/- 46, and 121 +/- 23, and with PMMA 63 +/- 13, 246 +/- 93, and 211 +/- 86, respectively. These results did not correlate with the effects of the membranes on complement activation. It is concluded that the release of cytokines during HD apparently leads to a rapid synthesis of acute-phase proteins as a sign of inflammation. Thus, SAA may be used as one indicator of the biocompatibility of HD treatment.

Effect of Endotoxin-induced Cell Injury on 70-kD Heat Shock Proteins in Bovine Lung Endothelial Cells

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.

Recombinant interleukin-1 stimulates clearance of Escherichia coli K1 bacteraemia

Enhancement of non-specific resistance to neonatal Escherichia coli K1 infection by inter-leukin-1 (IL-1) was analysed. Recombinant human IL-1 administered prophylactically to newborn LPS-non-responsive C3H/HeJ mice induced rapid clearance of E. coli 018:K1 bacteraemia. The effect was dose-dependent and was observed with mice treated immediately to 1 day before bacterial challenge, whereas treatment 2 days before challenge was ineffective. Clearance of intravenously injected radiolabelled 018:K1 E. coli suggested that IL-1 triggered defence mechanisms that contribute to bacterial sequestration and killing in the spleen and liver. Comparable increase in bacterial clearance occurred in naturally resistant LPS-responsive mice that had been subjected to transient E. coli K1 bacteraemia and showed increased resistance to reinfection. In the course of E. coli K1 bacteraemia a strong synthesis of acute phase reactants was observed in both susceptible and resistant mouse strains, which indicated that these proteins alone cannot confer natural resistance to E. coli K1. IL-1 induced a very rapid synthesis of acute phase proteins. The clearance of K1 E. coli when still viable in IL-1-treated animals suggested that acute phase proteins are not likely to be major mediators of the IL-1-enhanced non-specific resistance.

Biotechnology and Medicine of the Future

The practice of biology and medicine has been revolutionized during the past ten years by the advent of three biotechnologies--recombinant DNA techniques, the monoclonal antibody technology, and the development of microchemical instrumentation, machines that permit the rapid and effective synthesis and sequence analysis of proteins and genes. In this article, these powerful biotechnologies will be discussed, with particular emphasis on microchemical instrumentation, a major focus of my efforts for the past 15 years. I will also discuss two fundamental problems in modern medicine that are being explored in my laboratory using these techniques--genetic engineering of the nervous system and the mapping and sequencing of the human genome.

Distribution of insulin receptors between plasmalemma and intracellular compartments: Effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.

Protein synthesis requirement for acute ACTH stimulation of adrenal corticosteroidogenesis.

The rapid, cAMP mediated increase produced by ACTH in adrenal corticosteroidogenesis depends also on the rapid synthesis of protein. This obligatory involvement of protein synthesis has been established in several laboratories mainly on the basis of studies demonstrating a parallelism, under a variety of experimental conditions, of the capacity of adrenal cells to synthesize protein with their ability to increase steroid production in response to ACTH or cAMP. More recent correlative studies have disclosed the existence of two proteins, denoted as p and i. Protein i appears only in ACTH or cAMP stimulated cells and with the same time course and ACTH dose response as the increase in corticosteroid synthesis. Protein p, which closely resembles i in molecular weight and primary structure but differs in pI, is synthesized only in unstimulated cells. Neither the function of these two proteins nor the exact nature of the interplay of their synthesis on regulation has been established, although it seems reasonably certain that i is stimulatory rather than p inhibitory. The protein glycosylation inhibitor tunicamycin was found to inhibit the ACTH produced increase in steroidogenesis and also to inhibit the synthesis of protein i under conditions which did not inhibit overall protein synthesis. Therefore it seems probable that i is an N-glycosylated form of protein p and that adrenal cells are unresponsive to acute ACTH action if they are incapable of synthesizing glycosylated proteins specifically.

A Mg 2+ requirement for rapid ATP-driven protein synthesis by intact pea chloroplasts

Protein synthesis in the dark by chloroplasts isolated from young pea plants, driven by ATP, was slow unless Mg 2+ was added. Maximal stimulation occured when the added Mg 2+ was equimolar with the added ATP. The amount of incorporation of [ 3H] leucine into protein in the dark exceeded that found using light as the source of energy. When present in excess over the Mg 2+ level, ATP inhibited protein synthesis, largely by shortening the time course of the reaction. A low level of free Mg 2+ in the medium may have an important role in maintaining active protein synthesis by isolated pea chloroplasts. No indication was found of a need for light-activation of the enzymes of protein synthesis.