Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Salt stimulation of the activation of latent protein phosphatase, Fc.M, by Mn++ and by Mn/trypsin

The activation of porcine heart latent protein phosphatase (Fc.M) by pretreatment with Mn++ followed by trypsin (Mn/trypsin) can be stimulated 2.5-fold by including NaCl or KCl in the activation mixtures. The salts also stimulated the activation of the enzyme by Mn++ to the same level as that obtained by Mn/trypsin pretreatment in the absence of salt. The presence of salt in both the Mn++ and Mn/trypsin activations decreased the Mn++ requirement 10-fold in each case. Treatment of latent Fc.M by Mn/trypsin in the presence of 0.2 M NaCl or KCl offers a convenient method of expressing the full potential activity of the protein phosphatase.     

Latent forms of type-1 protein phosphatase in rabbit skeletal muscle

We have examined the characteristics of partially purified forms of rabbit skeletal muscle type-1 protein phosphatase (PP-1). Over 90% of PP-1 in unfractionated extracts and in glycogen particles was inactive, but could be activated by the divalent cations, Mn2+ or Co2+ (Me2+) plus trypsin. Gel filtration of muscle extracts revealed two inactive forms of PP-1; one activated by Me2+ alone or Me2+ plus trypsin, and a second containing inhibitor-2 and activated only by Me2+ plus trypsin. The kinetics of Me2+ plus trypsin activation revealed that after DEAE-chromatography, PP-1 was altered from the forms in crude extracts, gel filtered extracts or glycogen particles. The results indicate that the purified form of PP-1 catalytic subunit has properties which distinguish it from the forms of the enzyme in muscle extracts.     

Association of nascent polypeptide with 30S ribosomal subunits

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg²⁺ concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

Purification and properties of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) from Streptomyces antibioticus.

Two forms of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) have been purified from Streptomyces antibioticus. The larger form has an M(r) of 88,000, while the M(r) of a smaller form is 47,000. Both synthetase forms are active in the formation of guanosine 5'-triphosphate, 3'-diphosphate in reaction mixtures containing methanol. Unlike the RelA protein from Escherichia coli, the synthetases from S. antibioticus do not use GDP efficiently as a substrate. Experiments using crude extracts of S. antibioticus mycelium and the 88,000-M(r) form of guanosine pentaphosphate synthetase strongly suggest that the 47,000-M(r) species is produced by proteolysis of the larger species. This conclusion is supported by the observation that antibody to either protein reacts with the other protein. Thus, the 88,000-M(r) species may be the catalytically relevant protein in vivo. Unlike the RelA protein, the 88,000-M(r) protein is not activated by ribosomes. Modest levels of guanosine pentaphosphate synthesis were observed in mycelial extracts derived from nine other actinomycetes.     

Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.     

Serine proteinase activation of latent human skin collagenase

Latent and active collagenase were demonstrated following direct extraction from normal skin homogenates with 0.1M calcium chloride at 60 degrees C. 83% of the collagenase activity was in latent form and could be maximally activated with trypsin. Partial activation of the latent enzyme could also be demonstrated by incubation of the skin extract without added trypsin. This endogenous activation was inhibited by the addition of soya bean trypsin inhibitor, trasylol, di-isopropylphosphofluoridate and phenylmethanesulphonylfluoride, none of which inhibited collagenase directly. This suggests that the skin extracts contain a collagenase activating enzyme with the inhibition profile of a serine proteinase. A chymotryptic proteinase with a similar inhibition profile was extracted from normal human skin and partially purified. This enzyme activated fibroblast procollagenase derived from tissue culture of normal skin. The procollagenase was also partially activated by plasmin and chymotrypsin. This is the first demonstration of a collagenase activating enzyme in human skin and raises the possibility that collagenase activation by this mechanism may be responsible for collagen degradation in some disease processes.     

Activation of an insulin-stimulated S6 kinase in 3T3 L1 cell-free extracts by proteolysis

Using chromatography on a Fast S-Sepharose column, the insulin-stimulated S6 kinase can be resolved from other S6 kinases present in 3T3 L1 cell extracts. Only one S6 kinase is greatly activated by insulin (4-5-fold) and phosphorylates S6 with a high stoichiometry (4-5 mol phosphate per mol S6). This insulin-dependent S6 kinase can be activated in cell-free extracts by incubation with high concentrations of Ca2+. This activation is blocked by protease inhibitors such as leupeptin and is mimicked by trypsin. The stimulation does not require the presence of the protein kinase dependent on phospholipids and calcium (PK-C) in the cell extracts. The level of stimulation produced by proteolysis in the cell extracts is comparable to that reached in vivo by incubation with insulin.     

In vitro modification of bovine lens aldose reductase activity

Bovine lens aldose reductase can be activated in crude extracts upon incubation at 37 degrees C at relatively high ionic strength. This phenomenon shows a seasonal occurrence, the enzyme being susceptible to activation only in lenses of animals sacrified in summer. Systems generating oxygen activated species induce the enzyme activation, whereas scavengers of "oxygen radicals" preserve the activated state of the enzyme. Glutathione and other thiol compounds appear to prevent the enzyme activation.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase

The tryptophan hydroxylase activity of the crude extract from rat brain stem was stimulated approximately 2-fold by incubation with cAMP analogues under protein phosphorylating conditions. The cAMP-dependent activation process of the enzyme needed not only cAMP-dependent protein kinase but also activator protein. The kinetic properties of the enzyme activated by cAMP-dependent protein kinase were very similar to those of the enzyme activated by calmodulin-dependent protein kinase II.     

In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.     

Interactions of 5-lipoxygenase with membranes: studies on the association of soluble enzyme with membranes and alterations in enzyme activity

Treatment of rat basophilic leukemia cells (RBL-1) with the calcium ionophore A23187 resulted in activation of 5-lipoxygenase, as indicated by an induction of leukotriene release [Orning, L., Hammarstr?m, S., & Samuelsson, B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2017]. The enzyme activation was accompanied by a time-dependent association of 5-lipoxygenase to the particular fraction. When cells were lysed in the presence of 0.05-10 microM CaCl2, the soluble 5-lipoxygenase became associated with the particulate fraction. This was demonstrated by a decrease in immunoreactivities and enzymatic activities in the soluble fraction and a parallel increase in particulate-associated immunoreactivities. The particulate-bound enzyme was not active. Ca2+ induced the membrane association of 5-lipoxygenase when added into the incubation mixtures containing the membrane fraction with either the cytosolic fraction or the purified enzyme. 5-Lipoxygenase also bound to the microsomal-enriched fraction in the presence of Ca2+. Maximal membrane binding was obtained after a 1-min incubation at 4 degrees C. When a fixed amount of isolated membranes (0.2 mg of protein) and increasing cytosolic protein (0.5-4 mg) were used, a linear increase in enzyme binding was observed. The binding became saturated at 3 mg of cytosolic protein/mg of membrane protein. 5-Lipoxygenase binding to the membrane fraction was unaffected by pretreatment of the membranes with trypsin but was inhibited by treating with phospholipase A2, suggesting that phospholipids are involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of Mucor rouxii adenosine 3′:5′-monophosphate phosphodiesterase by phosphorylation-dephosphorylation and proteolysis

Partially purified cAMP phosphodiesterase from Mucor rouxii can be reversibly activated from 1.5- to 3-fold by treatment with MgATP, cAMP, and cAMP-dependent protein kinase, without change in its sedimentation behavior. Deactivation of activated enzyme can be observed in crude extracts under conditions which promote dephosphorylation; deactivation is prevented by 20 mm phosphate. cAMP phosphodiesterase can also be irreversibly activated by treatment with trypsin. The extent of activation by proteolysis is similar to that obtained by phosphorylation, but is accompanied by a decrease in the sedimentation coefficient of the enzyme. Activation by phosphorylation and proteolysis are not additive, suggesting that both mechanisms involve the same region of the enzyme molecule.     

Phosphorus-31 nuclear magnetic resonance of double- and triple-helical nucleic acids. Phosphorus-31 chemical shifts as a probe of phosphorus-oxygen ester bond torsional angles

The temperature dependence to the 31P NMR spectra of poly[d(GC)] . poly [d(GC)],d(GC)4, phenylalanine tRNA (yeast) and mixtures of poly(A) + oligo(U) is presented. The 31P NMR spectra of mixtures of complementary RNA and of the poly d(GC) self-complementary DNA provide torsional information on the phosphate ester conformation in the double, triple, and "Z" helix. The increasing downfield shift with temperature of the single-strand nucleic acids provides a measure of the change in the phosphate ester conformation in the single helix to coil conversion. A separate upfield peak (20-60% of the total phosphates) is observed at lower temperatures in the oligo(U) . poly(A) mixtures which is assigned to the double helix/triple helix. Proton NMR and UV spectra confirm the presence of the multistrand forms. The 31P chemical shift for the double helix/triple helix is 0.2-0.5 ppm upfield from the chemical shift for the single helix which in turn is 1.0 ppm upfield from the chemical shift for the random coil conformation.     

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.     

Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Dithiothreitol (DTT) has been found to stimulate the desacetoxycephalosporin C synthetase (expandase) activity of a frozen crude extract of Streptomyces clavuligerus, even in the presence of an optimum concentration of ascorbate. Catalase, both native and heat-denatured, and bovine serum albumin (BSA) also stimulated the enzyme activity but were less active than DTT. Although fresh extracts were sporadically stimulated, frozen extracts or extracts inactivated by shaking at 37°C in air were consistently activated. It is felt that DTT functions as a reactivating, rather than an activaing, agent. Similar effects were observed with extracts of Cephalosporium acremonium.     

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.