William H. Elliott

Characteristics of extracellular protease formation by Bacillus subtilis and its control by amino acid repression

Abstract 1. The formation of extracellular protease has been studied in washed-cell suspensions of Bacillus subtilis . Formation of the enzyme is inhibited by chloramphenicol and actinomycin D; its production is therefore due to de novo synthesis of the enzyme. 2. An examination of the distribution of protease between extra- and intracellular phases in washed cells showed the absence of any significant pool of preformed enzyme accumulating inside the cell over a 3-h period. 3. The rate of protease synthesis (but not of the simultaneously produced exoenzyme, α-amylase) is strongly repressed by the amino acids in the medium. No single amino acid is completely effective, but a mixture of either isoleucine and proline or of glutamic acid, aspartic acid, glutamine and asparagine gives maximal repression of protease formation.

Control of 5-aminolevulinate synthase in animals

The proposed mechanism by which hepatic ALV-synthase mitochondrial levels are regulated is outlined in Fig. 2. ALV-synthase catalyzes the first and rate-limiting step in the heme pathway and is normally present in low amounts. A cytosolic, regulatory free heme pool tightly controls the amount of ALV-synthase in two ways. In the primary mechanism of regulation, heme is proposed to inhibit the synthesis of ALV-synthase mRNA. Most likely this would be mediated through the action of specific heme-binding protein(s) which recognize regulatory control regions of the ALV-synthase gene. Gene activity therefore is significantly repressed most of the time. When there is an increased demand for heme by newly synthesized cellular hemoproteins, the free heme pool is reduced, leading to a derepression of ALV-synthase mRNA synthesis. Once the need for increased heme synthesis is satisfied, inhibitory heme levels build up again. When drugs such as phenobarbital are administered to animals, there is a rapid induction in the liver of both cytochrome P-450 and ALV-synthase. It is proposed that the heme pool governing ALV-synthase levels is lowered by the increased heme demand due to cytochrome P-450 apoprotein formation. The primary event in the drug induction of ALV-synthase is therefore the increased synthesis of cytochrome P-450 apoprotein. However, the mechanism by which this occurs is unknown, although drugs do increase the synthesis of mRNA for cytochrome P-450 (Fig. 2). (There is evidence that for the aromatic hydrocarbons a specific cytosolic receptor exists.) In the acute hepatic porphyria diseases, uncontrolled synthesis of hepatic ALV-synthase occurs. The various forms are characterized by reduced levels of one of the heme pathway enzymes other than ALV-synthase. Attacks of the disease are commonly precipitated by drugs which induce cytochrome P-450, and the uncontrolled accumulation of ALV-synthase which accompanies these attacks results from the combined action of the block in the heme pathway and the increased cytochrome P-450 levels. A major challenge which now exists is to understand at the molecular level how the genes for ALV-synthase and cytochrome P-450 are regulated in the liver and other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and properties of a specific bacterial ribonuclease inhibitor

Abstract 1. 1. Cultures of Bacillus subtilis cells produce extracellular ribonuclease towards the end of the growth phase. At the same time there is the production inside the cell of an inhibitor which is capable of inactivating the extracellular enzyme. 2. 2. The inhibitor can be selectively released by suddenly cooling the cells over a 14–16° temperature range. Such cold shocked cells lose only 4 % of total protein and show no gross morphological changes. Protoplast studies show the inhibitor to occur inside the cell membrane. 3. 3. The inhibitor has been purified and shown to have the properties of a protein of mol. wt. 12 500. Its properties are described. 4. 4. The inhibitor apparently forms a complex with the enzyme; inhibiton is not reversible by urea, salt, heating or sulphydryl reagents in contrast to the mammalian system. It has no action on pancreatic ribonuclease or ribonuclease T 1 . 5. 5. Using the paradoxical effect of actinomycin in stimulating ribonuclease synthesis in B. subtilis it has been shown that inhibitor and ribonuclease are independently synthesised and are not parts of a primary zymogen molecule. The possibility that the inhibitor is a secretory carrier for the enzyme has also been excluded.

Hemin inhibits transfer of pre-δ-aminolevulinate synthase into chick embryo liver mitochondria

Abstract Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-δ-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.

Dynamic state of the messenger RNA pool specific for extracellular protease in Bacillus amyloliquefaciens: Its relevance to the mechanism of enzyme secretion☆

Abstract It has previously been shown that Bacillus amyloliquefaciens possesses a large pool of messenger RNA capable of supporting extracellular protease synthesis for over 60 minutes. The breakdown of this pool of mRNA has been followed by taking the amount of rifampicin-insensitive protease synthesis to be an indication of the level of the mRNA. In the presence of high levels of amino acids known to repress protease synthesis, the pool disappears more rapidly than in low levels of amino acids. Amino acids appear to affect neither the translation nor the stability of the mRNA since in the presence of rifampicin the pool disappears at approximately the same rate in high and low amino acids. It is concluded that transcription of the protease mRNA is repressed by amino acids and that the pool is the product of a dynamic equilibrium between synthesis and degradation. The degradation of protease mRNA is not obligatorily dependent on continued protein synthesis. It is speculated that the excessive production of mRNA may be a mechanism to ensure that sufficient intact mRNA reaches membrane localised translational sites to support extracellular enzyme synthesis despite its rapid degradation. In this sense it may represent a primitive prokaryote messenger transport system.

Selective inhibition of extracellular enzyme synthesis by removal of cell wall from Bacillus subtilis

Abstract 1. 1. Protoplasts of Bacillus subtilis are rapidly lysed by l -glutamic acid and by a heat-stable factor present in the culture supernatant of older secreting cells. 2. 2. The formation of extracellular enzymes (α-amylase and protease) from such protoplasts has been compared with that from intact cells under identical conditions. Intact cells produce considerable amounts of these enzymes, but, by comparison, protoplasts fail to secrete significant amounts. 3. 3. Protoplasts incorporate [14C]valine and [14C]uracil into a trichloroacetic acid-precipitable form. The rate of [14C]valine incorporation is not significantly different from that by intact cells. However, [14C]uracil is incorporated by protoplasts at a rate which is considerably faster than that by intact cells. It therefore appears that removal of the cell wall specifically interferes with the synthesis of extracellular enzymes.

Evidence for a cytosolic precursor of chick embryo liver mitochondrial δ-aminolevulinate synthase

Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.

Purification of rat liver mitochondrial δ-aminolaevulinate synthase

Abstract Mitochondrial δ-aminolaevulinate synthase was purified from drug-induced adult rat liver and identified for the first time as a protein of minimum molecular weight 70,000. The enzyme was also identified in mitochondria by pulse-labelling and immunoprecipitation and shown to have a molecular weight of 70,000. The purified enzyme was degraded by papain to smaller forms of molecular weight about 56,000 with no loss of enzyme activity. In vitro translation experiments suggest that the enzyme is synthesized initially as a larger precursor of molecular weight 76,000.

cAMP-dependent induction of δ-aminolevulinate synthase in isolated embryonic chick liver cells

Abstract Isolated liver cells were prepared from 17-day old chick embryos and incubated in Eagles basal medium. Induction of δ-aminolevulinate synthase activity occurred immediately upon addition of allylisopropylacetamide and was totally dependent on the presence of Bt2cAMP (or cAMP) during the first 6 h of incubation. Under optimal inducing conditions in the presence of desferrioxamine mesylate and hormones, δ-aminolevulinate synthase induction occurred at rates comparable with those observed in ovo. The isolated liver cells provide a convenient experimental system for studying the effect of porphyrogenic drugs on porphyrin metabolism.

Cerulenin Inhibits Production of Extracellular Proteins but not Membrane Proteins in Bacillus amyloliquefaciens

Cerulenin inhibited the secretion of extracellular proteins by washed cell suspensions of Bacillus amyloliquefaciens. α-Amylase and protease secretion were inhibited by 80% and 75%, respectively, over 3 h. Cerulenin at 100 μg ml-1 inhibited incorporation of [1,2-14C]- acetate into intracellular lipid by about 75% without affecting cell growth, total protein synthesis or membrane protein synthesis. The inhibitory effect of cerulenin on α-amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens or various individual pure fatty acids. Cerulenin significantly altered the ratio of lipid to protein in isolated membranes. However, this alteration was not affected by adding fatty acids which restored enzyme secretion. These results suggest that cerulenin may affect the availability of lipid directly concerned with the secretion process. The differential effect of cerulenin on the production of extracellular proteins and membrane proteins also suggests that the synthesis of these two classes of proteins occurs via mechanisms that differ.