Characterization of the stimulatory effect of T4 gene 45 protein and the gene 4462 protein complex on DNA synthesis by T4 DNA polymerase

On a variety of single-stranded DNA templates, the overall rate of in vitro DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase is increased about fourfold by addition of the T4 gene $\text{44}\text{62}$ and 45 proteins. Several different methods suggest that this stimulation reflects an increase in the average DNA polymerase “sticking distance”, or processivity, from 800 to about 3000 nucleotides per initiation event. Both the $\text{44}\text{62}$ protein complex and the 45 protein must be present to obtain this effect, and either ATP or dATP hydrolysis is required. Rapid-mixing experiments indicate that the polymerase stimulation is maximized within a few seconds after addition of these “polymerase accessory proteins.”     

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro

Preovulatory follicles isolated from immature rats, treated $\text{in vivo}$ with pregnant mare's serum gonadotropin, were incubated $\text{in vitro}$ and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

The acid-base properties and kinetics of dissolution of the Fe4S4 cores of Chromatin ferredoxin and high potential iron protein.

From a study of the translation of synthetic polynucleotides in the $\text{E. coli}$ and $\text{B. thuringiensis}$ cell-free systems, it is shown that the stimulation of polypeptide synthesis by spermidine depends on the uracil content of messenger ribonucleic acid. This stimulation can not be fulfilled by any amount of Mg²⁺ in the absence of polyamines.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

Role of NAD+ in the stimulation of protein synthesis in rabbit reticulocyte lysates.

The stimulation of protein synthesis by NAD⁺ in rabbit reticulocyte lysates has been reported. [Lennon M. B., Wu, J., and Suhadolnik, R. J., (1976) Biochem. Biophys. Res. Commun. 72, 530–538]. NAD⁺ can replace the creatine phosphate-creatine phosphokinase ($\text{CP}\text{CPK}$) energy regenerating system normally used in in vitro protein synthesizing systems. The replacement of $\text{CP}\text{CPK}$ by NAD⁺ is optimal at 37 °C. A significant lag in the rate of protein synthesis with NAD⁺ is observed with decreasing temperatures. Analysis of the adenylate energy charge with NAD⁺ shows an initial rapid decrease. This decrease in the energy charge recovers with increasing NAD⁺ concentrations. The energy level correlates with the rates of incorporation of d,l-[4,5-³H(N)]leucine into protein. ATP production via NAD⁺ pyrophosphorylase or oxidative phosphorylation does not explain the stimulation by NAD⁺. Rather, the stimulation is correlated with the activation of glycolysis. Glycolysis is not active in lysate preparations because NAD⁺ is absent. Additional possible roles of NAD⁺ in protein synthesis are discussed.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Responsibility of 16S RNA for the stimulation of polypeptide synthesis by spermidine.

From the studies on the spermidine stimulation of polyphenylalanine synthesis catalyzed by $\text{E. coli}$ 50S and reconstituted 30S particles containing 16S RNA and 30S ribosomal proteins from $\text{E. coli}$ and $\text{B. thuringiensis}$ in different kinds of combinations, it is concluded that 16S RNA is mainly responsible for the stimulation of polypeptide synthesis by spermidine.     

Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems.

In the presence of polyamines, the fidelity of protein synthesis in a wheat germ cell-free system was increased significantly, while it was increased slightly in an $\text{E. coli}$ cell-free system. The effective concentration of polyamines for the increase in fidelity of protein synthesis was nearly equal to that for the stimulation of protein synthesis in a wheat germ cell-free system.     

Effect of carbamyl phosphate on the regulation of nitrogenase in Clostridium pasteurianum

One mM carbamyl phosphate inhibited the $\text{in}$$\text{vitro}$ acetylene reduction activity of nitrogenase 30% whereas at high concentrations a maximum inhibition of 50% was observed. When 1 mM carbamyl phosphate was added to a culture growing of N₂ 1) nitrogenase synthesis was completely repressed and 2) after a period of 2.5 hrs in the absence of growth, the specific activity decreased to less than 50% of its activity just before the addition of the inhibitor.     

Pyruvate and nitrogenase activity in cell-free extracts of the blue-green alga Anabaena cylindrica

When extracts of $\text{Anabaena cylindrica}$ are prepared in the absence of dithionite, they catalyze pyruvate-dependent acetylene reduction, a reaction not observable in assays containing dithionite. Ferredoxin and coenzyme-A, but not NADP and ferredoxin-NADP reductase, are required for maximal pyruvate-dependent activity. These acetylene-reducing extracts do not exhibit NADP-pyruvate dehydrogenase activity. However, pyruvate:ferredoxin oxidoreductase is present at levels of activity sufficient to support the $\text{in vitro}$ rate of pyruvate-supported acetylene reduction. These $\text{in vitro}$ data support earlier $\text{in vivo}$ evidence that pyruvate:ferredoxin oxidoreductase transfers electrons from pyruvate to nitrogenase in $\text{A. cylindrica}$.     

Regulation of the basal and cyclic AMP-stimulated rates of glycogen synthesis in Escherichia coli by an intermediate of purine biosynthesis

In $\text{Escherichia}\text{coli}$an abrupt increase in the rate of glycogen synthesis occurs at the onset of total nitrogen starvation. We present here both $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$ data indicating that this increase occurs because of the loss of a nitrogen-containing intermediate of purine biosynthesis (apparently 5-aminoimidazole-4-carboxamide ribonucleotide) that inhibits glycogen synthesis. We also show that this inhibitory intermediate antagonizes the stimulation of glycogen synthesis by 3′,5′-cyclic AMP. The uncovering of the regulation of glycogen synthesis by this inhibitor apparently provides the first link in understanding the 23-year-old observation of a reciprocal relationship between growth rate and glycogen accumulation in $\text{E.}\text{coli}$.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

The receptor-mediated, short-term activation of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia.

Tyrosine hydroxylation, measured $\text{in situ}$ in superior cervical ganglia from neonatal rats, is increased two-fold by the addition of carbachol. This increase occurs within one hour and is not due to the synthesis of new enzyme protein, nor to a direct effect of carbachol on the enzyme. The stimulation is blocked by atropine, but not by hexamethonium. This stimulation represents the first report of a receptor-mediated short-term action on this rate-limiting enzyme.     

Pyruvate-supported acetylene and sulfate reduction by cell-free extracts of Desulfovibrio desulfricans

Cell-free extracts prepared from a strain of $\text{Desulfovibrio}\text{desulfricans}$ can reduce acetylene or sulfate, utilizing pyruvate as the electron and ATP source in the presence of methyl viologen, ADP and coenzyme A. Other physiological substances such as (lactate + NAD) and (NADH + ATP) can not reduce acetylene nor sulfate. When acetylene and sulfate are both present as substrates, sulfate represses the acetylene reduction.     

Effects of 6-thioguanine-containing RNA on in vitro protein synthesis

6-Thioguanine was administered to rats 12 hr after partial hepatectomy at a dose of 40 mg/kg of body weight; 6 hr later, polyadenylic acid-containing RNA was isolated and was used to measure initiation of protein synthesis $\text{in vitro}$ in a wheat germ system. $\text{In vitro}$ initiation was found to be 2.3-fold greater when 6-thioguanine-containing RNA was employed, than when polyadenylic acid-containing RNA isolated from untreated animals was used. The homopolymer, poly(TG), did not promote peptide synthesis in the wheat germ $\text{in vitro}$ system employed.     

Mechanism of corticotropin action in rat adrenal cells I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis

The contribution of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steriodogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidegenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidegenesis but not on protein synthesis. The reversal effects was not observed at high levels of inhibitors. One inhibitor of microfilament fromation (cytochalasin B) and four inhinitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhinitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–6}\text{min}$). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3–5 min, $\text{f}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7–9}\text{min}$). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhbition of cytochrome P-450_sec by an aminoglutethimide was complete within 1 min and was rapidly reversed.Injection of each inhibitors (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450_sec in adrenal mitochondria isolated from these rats was also decreased by all inhbitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450_sec (r = 0.94).It is concluded that protein synthesis and steroidogenesis must be intimately coupled propbably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450_sec. An involvement of microtubules and microfilaments in this process is clearly indicated.     

The effect of beta-naphthoflavone on rabbit liver protein synthesis, and on the induction of cytochrome P-450 LM4 mRNA

A single injection of β-naphthoflavone dispersed in corn oil causes significant changes in rabbit liver polysome and polysomal poly(A+)mRNA driven $\text{in vitro}$ protein synthesis. The changes occur between 6–18 hr and 30–36 hr after the injection. Our data indicate that the first effect is due to the β-naphthoflavone and the second effect is due to the oil vehicle. $\text{In vitro}$ translation of rabbit liver polysomes obtained from treated rabbits followed by specific immunoprecipition and gel electrophoresis, showed that maximal levels of translatable cytochrome P-450 LM₄ occurred 18–24 hr after β-naphthoflavone treatment.     

Exogenous methionine depresses level of mRNA for a soybean storage protein

$\text{In vitro}$ translation experiments indicate that absence of the β-subunit of 7S storage protein in soybean ($\text{Glycine max}$ L. Merr. cv. Provar) cotyledons cultured on methionine-supplemented medium is due to lack of functional mRNA for that polypeptide. Relative amounts of functional mRNA for the 7S α′- and α-subunits were unaffected by methionine in the cotyledon culture medium. Measurements of β-subunit accumulation in cotyledons transferred from basal medium to methionine-supplemented medium show that methionine inhibits continued accumulation of the β-subunit after synthesis of the β-subunit has begun, and that methionine does not promote degradation of existing β-subunit.     

Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon.

The regulation of the $\text{in vitro}$ synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacp^s or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of $\text{lac}$ repressor in the $\text{E. coli}$ S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)¹ for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacp^s DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen.