Edward Herbert

Factors affecting the rate of protein synthesis in lysate systems from reticulocytes

Abstract The addition of hemin to a reticulocyte lysate system stimulates protein synthesis under a variety of incubation conditions. When an optimal concentration of Mg2+ (2mm) is employed and creatine phosphate and creatine phosphokinase are used as the ATP regenerating system, incorporation of valine occurs at better than 60% of the rate in the intact cell for 30 minutes at 28 ° in the presence of added hemin. The main effect of hemin is to sustain the activity of the system rather than to increase the rate of incorporation. The concentration of hemin that produces maximal stimulation of protein synthesis is between 5 × 10−5 and 1 × 10−4 m . Deuterohemin stimulates incorporation to a lesser extent than hemin but in the same concentration range. After 10 minutes preincubation at 28 ° the capacity of the lysate to respond to hemin is lost, whereas the capacity to synthesize protein is reduced by less than 50%. Exogenous hemin increases the extent of de novo synthesis of hemoglobin and, at 28 °, greatly retards disaggregation of polysomes that accompanies protein synthesis in the lysate. The effect of hemin is similar to that observed in the intact cell.

Acetylcholine and lobster sensory neurones

Experiments are presented in support of the hypothesis that acetylcholine functions as a sensory transmitter in the lobster nervous system.

Effects of cycloheximide on polyribosome function in reticulocytes.

Abstract 4 × 10 −6 m -Cycloheximide partially inhibited protein synthesis in reticulocytes, and caused a slight increase in the polyribosome content of the cells and the amount of nascent polypeptide material bound to polyribosomes. When reticulocytes were incubated with NaF, with chloramphenicol, or without added iron salts, polyribosomes were converted to single ribosomes. This conversion could be prevented by the addition of 4 × 10 −6 m -cycloheximide. When cycloheximide was added after polyribosome breakdown had taken place, the polyribosomes were re-formed. Re-formation of polyribosomes was accompanied by globin synthesis, and the polyribosomes formed carried nascent polypeptide chains. A higher concentration of cycloheximide (10 −4 m ) caused breakdown of the polyribosomes during the first 20 minutes of incubation. However, polyribosomes breakdown did not take place when reticulocytes were incubated for 50 minutes with 1.5 × 10 −2 m -cycloheximide. 1.5 × 10 −2 m -cycloheximide prevented the breakdown of polyribosomes in cells incubated with NaF but did not cause polyribosome formation when added after breakdown had taken place. In reticulocytes incubated with 1.5 × 10 −2 m -cycloheximide, there was no detectable incorporation of amino acids into soluble protein or nascent polypeptide chains during 60 minutes of incubation. The results are consistent with the hypothesis that protein synthesis is accompanied by an exchange of ribosomes between the polyribosomal aggregates and the pool of single ribosomes, and that the detachment of ribosomes from the aggregates is slowed by 4 × 10 −6 m -cycloheximide and strongly inhibited by 1.5 × 10 −2 m -cycloheximide.

Peptides in the nervous system

binding sites of G proteins with those of the products of the ras oncogenes will provide an impetus to such research. The development of antibody and oligonucleotide probes will permit exploration of potential alterations of receptors, G proteins, and effectors as a source of pathological function. Elucidation of a genetic deficiency of Gs~ as the cause of one type of pseudohypoparathyroidism is a start. It would be surprising if this interesting discovery was less than that. Selected references

Involvement of hemin, a stimulatory fraction from ribosomes and a protein synthesis inhibitor in the regulation of hemoglobin synthesis

Abstract For maximum globin synthesis in lysate cell-free systems prepared from rabbit reticulocytes, there is a requirement for both hemin and a fraction derived from the ribosome by salt treatment (I fraction). A previously described inhibitor of protein synthesis (Q fraction), which accumulates during incubation of the lysate system, is shown to be without effect on completion and release of nascent chains, suggesting that it is acting at the site of chain initiation. Inhibition of globin synthesis by Q fraction is not reversed by addition of salt-treated ribosomes but is blocked and reversed by the addition of the I fraction, suggesting that an I fraction component is limiting in systems containing Q fraction. Several modes of regulation of protein synthesis involving hemin, I fraction and Q fraction are discussed in the light of kinetic data obtained with these systems, and in the light of the finding that intact cells incubated in the absence of added iron accumulate an inhibitor with properties similar to Q fraction desscribed above.

Discovery of pro-opiomelanocortin-a cellular polyprotein

Abstract Pro-opiomelanocortin (POMC) contains the sequences of ACTH, -endorphin and melanocyte stimulating hormones. POMC is present in both the anterior and intermediate lobes of the pituitary but it is processed to different hormones in each lobe. Release of these hormones is also regulated differently in the two lobes. The various ways in which these differences in processing and regulation might arise in the two lobes of the pituitary are discussed.

Biosynthesis of polyprotein precursors to regulatory peptides

In the past 15 years a number of small peptides that mediate specific kinds of behavior in animals has been discovered. For example, hypothalamic releasing factors ranging from 3 to 41 amino acids regulate the release of anterior pituitary hormones that mediate reproductive cycles, stress responses and diurnal temperature and activity cycles. Substance P is a neurotransmitter in the pain pathways of animals. Opioid peptides, P-endorphin, enkephalins and dynorphin raise the threshold to pain. Other peptides have been implicated in sleep and the regulation of temperature (bombesin). Finally, small peptide pheromones (a and 01 factors) mediate conjugation in yeast. Biosynthetic studies have revealed that regulatory peptides are derived from proteins that are often 10 times or more the size of the biologically active peptide. Why has nature made such large proteins for the generation of such small peptides? For one thing, the peptides must be cut out of the precursor by proteolytic enzymes and be chemically modified to become active and to be secreted. Chemical modifications such as glycosylation, phosphorylation and methylation occur in a well defined order as newly synthesized secretory proteins move through the endoplasmic reticulum, Golgi complex and vesicles on their way to being secreted. Amino acid sequences specify the sites of modification and cleavage. For example, vectorial transport of a secretory protein across membranes of the endoplasmic reticulum is specified by a signal sequence of 12-30 amino acids (Blobel and Dobberstein, JCB 67, 852-862, 1975). Glycosylation of a protein at asparagine residues requires the sequence Asn-XXX-ihh,. Other modifications such as phosphorylation and methylation may activate or inactivate a peptide or expose or mask a cleavage site. Portions of the precursor sequence may therefore be necessary for specifying interactions involved in compartmentalization of proteins and their activation during secretion. In the case of proinsulin, the C peptide region assists in the formation of correct disulfide bridges in the insulin molecule, and quite likely serves as a spacer to elongate the peptide to a minimum length (probably 65-70 residues) for efficient membrane transport in the endoplasmic reticulum (Steiner, NY Acad. Sci. 343, l-l 6, 1980). Another reason for large precursors has become apparent in the past 5 years, namely, that they may contain sequences of several different biologically active peptides or multiple copies of the same peptide. Thus the precursor to adrenocorticotropic hormone

Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system.

Abstract In the absence of added hemin, the rate of protein synthesis in a lysate cell-free system from rabbit reticulocytes falls almost to 0 after 15 minutes incubation at 35 °C and the proportion of ribosomes present as polyribosomes falls from 50 to about 15%. Addition of hemin at this time restores protein synthesis to 60 to 70% of the initial rate in the presence of added hemin, and polyribosomes to almost 80% of the initial level (40% ribosomes as polyribosomes). If hemin is added before the start of incubation, a high rate of protein synthesis is sustained well beyond 20 minutes and the conversion of polyribosomes to monoribosomes is greatly retarded or prevented. Metal derivatives of protoporphyrin IX have been synthesized and tested for ability to replace hemin in sustaining the rate of protein synthesis in the intact cell and in the lysate system. Aggregation of these compounds in the solution added to the lysate has been found greatly to influence their effectiveness in this respect. When care is taken to reduce aggregation, it is found that the cobalt, nickel, magnesium and zinc derivatives will replace hemin in the lysate system, whereas the copper derivative will not. Fe 2+ and Co 2+ will not replace hemin in this system. It has been shown previously that the latter ions do replace hemin in the intact cell. Cobalt protoporphyrin IX and protoporphyrin IX sustain protein synthesis in the intact cell as well as Fe 2+ or hemin. Magnesium and zinc protoporphyrin IX derivatives are about half as effective as Fe 2+ in sustaining protein synthesis, whereas the copper and nickel derivatives are not effective at all in this respect. Stimulation of protein synthesis by hemin is prevented by the addition of Pb 2+ to the lysate system. Concentrations of Pb 2+ that prevent the hemin effect also strongly promote aggregation of hemin.

Immunohistochemical Demonstration of proGnRH and GnRH in the Preoptic-Basal Hypothalamus of the Primate

An antiserum (ARK-1) specific to the gonadotropin-releasing hormone precursor (proGnRH) was produced by immunizing with a synthetic peptide (proGnRH 6-16; Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-Asp-Ala-Glu) which bridges the proteolytic cleavage site of proGnRH. When used in the radioimmunoassay, ARK-1 bound 25% of the iodinated 5-16 fragment at a 1:30,000 dilution with a sensitivity of 1 pg/tube. Using immunohistochemical techniques, we observed that in serial and the same sections through the preoptic-basal hypothalamus (POA-BH), the precursor molecule was primarily present in the cell soma, whereas GnRH was found in the cell soma, nerve fibers, and terminals of the same neurons. These data indicate that the processing of proGnRH to biologically active peptides (e.g., GnRH) in the rhesus macaque and the baboon POA-BH primarily occurs in the cell soma.

The effect of chloramphenicol in intact erythroid cells.

Incubation of intact reticulocytes with chloramphenicol at concentrations over 10(-3) M caused inhibition of protein synthesis and conversion of polyribosomes to single ribosomes. At 3 x 10(-3) M-chloramphenicol these effects were completely reversible, but reversibility decreased as the chloramphenicol concentration was raised above this level. 3 x 10(-3) M-chloramphenicol also caused a 15% decrease in the ATP content of reticulocytes. Evidence was obtained that the intracellular concentration of chloramphenicol in reticulocytes was approximately equal to the concentration in the incubation medium. L(+)threo chloramphenicol inhibited protein synthesis in reticulocytes, and chloramphenicol inhibited the synthesis of both hemoglobin and RNA in bone marrow cells. These results indicate that the action of chloramphenicol in these cells is different from that in bacteria.