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P Setlow

Publications and source records attributed to P Setlow.

At least 217 records · Page 12Linked to original sources
PubMed1978

Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores.

Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.

Amino Acids↗
PubMed1978

Levels of cyclic GMP in dormant, germinated, and outgrowing spores and growing and sporulating cells of Bacillus megaterium.

The level of cyclic GMP was less than one molecule per organism in dormant, germinated, and outgrowing spores of Bacillus megaterium. A significant level (approximately 8 pmol/g, dry weight) of cyclic GMP was found in early to mid-log phase cells, but the level fell to below 0.2 pmol/g, dry weight, in late-log phase and only rose slightly to approximately 0.9 pmol/g, dry weight, in stationary phare. No significant amount of cyclic GMP was detected in the growth medium at any time.

Bacillus megaterium↗
PubMed1977

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.

Adenine Nucleotides↗
PubMed1977

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.

Bacillus megaterium↗
PubMed1977

Levels of small molecules and enzymes in the mother cell compartment and the forespore of sporulating Bacillus megaterium.

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.

Adenine Nucleotides↗
PubMed1977

Levels of acetyl coenzyme A, reduced and oxidized coenzyme A, and coenzyme A in disulfide linkage to protein in dormant and germinated spores and growing and sporulating cells of Bacillus megaterium.

Dormant spores of Bacillus megaterium were found to contain approximately 850 pmol of coenzyme A (CoA) per milligram of dry weight. Of this total, less than 1.5% was acetyl-CoA, 25% was CoA-disulfide, 43% was in disulfide linkage to protein, and the remainder was the free thiol. Dormand spores of Bacillus cereus and Clostridium bifermentans contained 700 and 600 pmol of CoA per milligram of dry weight, respectively; in both species approximately 45% of the CoA 45% of the CoA was in disulfide linkage to protein. During germination of spores of all three species, greater than 75% of the CoA-protein disulfides were cleaved. In B. megaterium, cleavage of these disulfides during spore germination did not require exogenous metabolites and occurred at about the same time as the initiation of germination. Much of the CoA was converted to acetyl-CoA at this time. Dormant spores also contained reduced nicotinamide adenine dinucleotide-dependent CoA-disulfide reductase at levels higher than those in other stages of growth. The level of total CoA in the growing cells was two- to three-fold higher than in spores. This level remained constant throughout growth and sporulation, but less than 2% of the total cellular CoA was in disulfide linkage to protein until late in sporulation. The CoA-protein disulfides accumulated exclusively within the developing spore at about the time when dipicolinic acid was accumulated.

Acetyl Coenzyme A↗
PubMed1977

Production of large amounts of acetate during germination of Bacillus megaterium spores in the absence of exogenous carbon sources.

When Bacillus megaterium spores germinate in the absence of an exogenous carbon source, the first minutes of germination are accompanied by production of large amounts (approximately 70 nmol/mg of dry spores) of acetate and much smaller amounts of pyruvate and lactate. The majority of these compounds are excreted into the medium. Exogenous pyruvate and alanine are also converted to CO2 and acetate by germinating spores, presumably by using the pyruvate dehydrogenase that is present in dormant spores. These data suggest that the 3-phosphoglyceric acid stores in the dormant spore and alanine generated by proteolysis early in germination can be catabolized to acetate during germination with production of large amounts of reduced nicotinamide adenine dinucleotide, acetyl coenzyme A, and adenosine 5'-triphosphate.

Acetates↗
PubMed1976

Purification and properties of a specific proteolytic enzyme present in spores of Bacillus magaterium.

A proteolytic enzyme with high activity on the specific, low molecular weight dormant spore proteins (termed A and B proteins) degraded during spore germination has been purified approximately 1000-fold from Bacillus megaterium spores. This enzyme accounts for greater than 85% of the proteolytic activity toward the A and B proteins in crude spore extracts. The protease has a pH optimum of approximately 7.5, is inactivated by phenylmethylsulfonyl fluoride and EDTA (10 mM), and is inhibited approximately 70% by NaCl (1 M). The enzyme is unstable and requires glycerol, divalent cations, and high enzyme concentrations for maximum stability. The protease shows a high degree of specificity for the A and B proteins, since high levels of enzyme catalyze no detectable bond cleavage on a variety of amide, ester, peptide, or other protein substrates. The enzyme is an endoprotease and digestion of the A and B proteins in vitro generates a number of peptide fragments at least one of which appears identical with a fragment isolated from lysates of spores carrying out hydrolysis of endogenous A and B proteins. In vitro, the peptide products can be rapidly degraded to amino acids by an aminopeptidase which has also been partially purified from spores. Although high levels of the protease are present in extracts of dormant spores, and of spores germinated for a few minutes, the enzyme is undetectable in log phase and early stationary phase cells. Furthermore, the protease disappears rapidly (t1/2 approximately 30 min) and completely (greater than 90%) as the process of germination proceeds.

Aminopeptidases↗
PubMed1975

Identification and localization of the major proteins degraded during germination of Bacillus megaterium spores.

Of the 15 to 20% of total spore protein which is degraded during germination of spores of Bacillus megaterium, greater than 80% is derived from a protein fraction soluble in dilute acids such as acetic acid that contains two major proteins, termed A and B. These two proteins are low or absent in germinated spores, log phase cells, and early stationary phase cells, but both are synthesized and appear in parallel late in sporulation after the appearance of nonrefractile forespores but before the appearance of refractile spores. These proteins accumulate during the time that the developing spore becomes resistant to ultraviolet light but well before acquisition of heat resistance. Both proteins are located in the core of the dormant spore, but analysis of ruptured spores has revealed no binding of protein A or B to ribosomes, membranes, or DNA.

Bacillus megaterium↗
PubMed1975

Purification and properties of some unique low molecular weight basic proteins degraded during germination of Bacillus megaterium spores.

Two major proteins, termed proteins A and B, and one minor species, termed protein C, have been purified to homogeneity from dilute acid extracts of dormant spores of Bacillus megaterium. These three species comprise approximately 80% of the protein in the dilute acid extracts and account for 60 to 75% of the protein degraded during spore germination. All three proteins have low molecular weights (7,000 to 10,000), high isoelectric points (greater than 9.8), alanine as the NH2-terminal amino acid, are more hydrophilic than most proteins, and all lack cysteine, cystine, and tryptophan. In addition all three proteins are extremely sensitive to a wide variety of proteolytic enzymes, much more so than "average" proteins such as serum albumin, lysozyme, and hemoglobin. These proteins also bind to both purified DNA and to a nuclear body from dormant spores. Although this binding gives little or no protection to proteins A and B from proteolysis, it does result in elevation of the melting temperature of the DNA by as much as 20degrees.

Amino Acids↗
PubMed1975

Protein metabolism during germination of Bacillus megaterium spores. I. Protein synthesis and amino acid metabolism.

Protein synthesis during germination of Bacillus megayerium spores can be divided into two stages. During the first 75 min of germination (Stage I) endogenous nitrogen reserves are sufficient to support protein synthesis, and most amino acids are generated by proteolysis of dormant spore protein. The amino acids produced are excreted initially from the spore, but then reabsorbed and partially utilized for protein synthesis. Significant amino acid metabolism also occurs during Stage I, utilizing enzymes already present in the dormant spore. The biosynthesis of a number of amino acids is low or absent during Stage I due to the absence of biosynthetic enzymes. Subsequently, at defined times in Stage I, these missing enzymes are synthesized and amino acid biosynthesis is initiated. By the beginning of Stage II (from 75 min on) the developing spore has regained the capacity for synthesis of all amino acids and requires an exogenous nitrogen source for rapid protein synthesis.

Amino Acids↗
PubMed1975

Protein metabolism during germination of Bacillus megaterium spores. II. Degradation of pre-existing and newly synthesized protein.

Two distinct proteolytic systems have been detected during germination of Bacillus megaterium spores: one degrading a unique class of dormant spore proteins and the other degrading primarily protein synthesized during germination. Proteolysis of dormant spore protein began by the 3rd min of germination and by 25 min had degraded 15 to 20% of the pre-existing protein to free amino acids. This reaction was not significantly ( less than 20%) different with or without amino acids or a carbon or nitrogen source in the germination medium, or when RNA synthesis, protein synthesis, or energy metabolism were inhibited. Spore coat proteins and most enzymes were not degraded in this process, rather the major substrates were a unique class of low molecular weight (6,000 to 12,000) proteins which were soluble in acetic acid. Proteins synthesized early in germination (0 to 12 min) were also degraded rapidly (20% per hour). However, proteins synthesized later in germination (90 to 100 min) were degraded more slowly (similar to 4% per hour). At all times tested proteolysis of newly synthesized protein was identical in the presence or absence of amino acids or chloramphenical in the medium, but was abolished by inhibitors of energy metabolism. Most proteins degraded in this process had molecular weights greater than 12,000 and were insoluble in acetic acid.

Amino Acids↗
PubMed1975

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.

Albumins↗