Kodaistatins, novel inhibitors of glucose-6-phosphate translocase T1 from Aspergillus terreus thom DSM 11247. Isolation and structural elucidation.

Two novel compounds, kodaistatin A, C35H34O11, molecular weight 630, and kodaistatin C, C35H34O12, molecular weight 646, have been isolated from cultures of Aspergillus terreus Thom DSM 11247 by solid-phase extraction, size-exclusion chromatography, and various preparative HPLC steps. The use of a range of 2D NMR measurements, in particular 13C-13C correlation measurements, has led to the clarification of the structure of kodaistatin A. Kodaistatin C is a hydroxylated derivative of kodaistatin A. Both natural products contain hydroxylated aspulvinones and identical highly substituted polyketide units. An X-ray single crystal structure analysis of aspulvinon E demonstrated the z-configuration at the central double bond. The kodaistatins are effective inhibitors of the glucose-6-phosphate translocase component of the glucose-6-phosphatase system (EC 3.1.3.9), an enzyme system which is important for the control of blood glucose levels. The IC50 is 80 nM for kodaistatin A and 130 nM for kodaistatin C.

Filarial parasites exhibit unusually high levels of choline acetyltransferase activity.

The presence of unusually high levels of choline acetyltransferase (ChAT, EC 2.3.1.6) in human and animal filarial parasites has been demonstrated. The levels of ChAT were highest in male worms of Brugia malayi and Brugia pahangi, with specific activities in crude extracts of about 2.27 and 1.26 mumol min-1 (mg protein)-1, respectively. The enzyme levels in these worms were over 10-20 times higher than in male worms of Litomosoides carinii. The ChAT levels were about 2-5 times higher in male than in female worms. The enzyme was also present in appreciably high levels in microfilariae of Brugia species, L. carinii and Wuchereria bancrofti. The levels of ChAT in male worms of Brugia species were several thousand-fold higher than in the intestinal nematodes Trichuris muris and Necator americanus, and were over three orders of magnitude higher than in mammalian brain. Unlike the mammalian ChAT, the parasite enzyme was extremely stable. The parasite enzyme was not inhibited by any of the antifilarial agents except suramin. The filarial ChAT was strongly inhibited by sulphydryl reagents and diethylpyrocarbonate. Ethacrynic acid (EA), a diuretic and a sulphydryl reagent, irreversibly inhibited the filarial ChAT activity at low concentrations. In contrast, EA inhibited the activity of mammalian brain ChAT at much higher concentrations. The motility of adult worms and microfilariae was irreversibly inhibited by low concentrations of EA. Furthermore, the inhibition of motility was paralleled by the inactivation of ChAT in these parasites. These studies indicate that ChAT activity appears to be vital for parasite's survival and that acetylcholine might play a key role in the control of worm motility.

Isolation, partial purification and some properties of two acid proteases from adult Dirofilaria immitis.

Two acid proteases were isolated from the soluble extracts of adult Dirofilaria immitis, the filarial heartworm of canines. Activity of these proteases was detected using 3H-labeled bovine alpha-casein as substrate, and they were designated Fp-I and Fp-II in order of their elution from a CM-cellulose column. The molecular weight of partially purified Fp-I was approximately 170000, and it was active between pH 4.6-5.8. The activity of Fp-I doubled in the presence of various sulfhydryl reagents at 5 mM, and it was inhibited 50-60% by the sulfhydryl inhibitors p-hydroxymercuribenzoate and iodoacetate at 1 mM, the heavy metal chelating agent o-phenanthroline at 1 mM and the peptide aldehyde protease inhibitors pepstatin (10 microM), leupeptin, antipain and chymostatin (50 microM). The molecular weight of the more extensively purified Fp-II is approximately 48000. This protease was active between pH 2.6-3.4 and was highly sensitive to inhibition by pepstatin (80% inhibition at 10 nM). Fp-II was not significantly affected by sulfhydryl reagents, sulfhydryl inhibitors, metal chelating agents or peptide aldehyde protease inhibitors other than pepstatin. These properties of dirofilarial Fp-II resemble those of mammalian cathepsin D.

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits.

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.

Subcellular distribution of various proteases in Escherichia coli.

It has been reported recently that Escherichia coli cells contain eight distinct soluble enzymes capable of degrading proteins to acid-soluble material. Two are metalloproteases that degrade [125I]insulin but not larger proteins: protease Pi, which is identical to protease III, is restricted to the periplasm, and protease Ci is restriction to the cytoplasm. The six others (named Do, Re, Mi, Fa, So, and La, which is the ATP-dependent protease) are serine proteases that degrade [14C]globin and [3H]casein, but not insulin. One of these (Mi) is localized to the periplasm, and one (Re) is distributed equally between the two cellular fractions. The others are present only in the cytoplasm.

Studies of the ATP dependence of protein degradation in cells and cell extracts.

Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation requires the continuous production of ATP. We have established soluble cell-free preparations from rabbit reticulocytes, rat liver, and Escherichia coli that degrade abnormal protein in an ATP-dependent fashion. These enzymes appear to be responsible for the selective breakdown of abnormal protein that may result from mutations, biosynthetic errors or intracellular denaturation. Experiments with inhibitors indicate that this process and the degradation of many short-lived normal proteins does not occur in the lysosome. The cell-free extracts prepared from these crude extracts hydrolyse [14C] globin by a process stimulated 2--3-fold by ATP and to a lesser extent by GTP, CTP or UTP. These activities degrade globin to large peptides which are then cleaved by soluble peptidases. The ATP-stimulated protease that partially purified from rat liver cytoplasm is also stimulated by pyrophosphate. This protease has an apparent molecular weight of 480,000. In contrast, the E. coli enzyme has an apparent molecular weight of 115,000 and is completely dependent on ATP, after partial purification by ion exchange and gel chromatography. This enzyme can be distinguished from six other proteolytic enzymes from E. coli active at pH 7.8. E. coli contains, in addition, four proteases that are not stimulated by ATP and degrade globin to acid-soluble material. We have also demonstrated in E. coli and reticulocytes other proteases that appear specific for small protein substrates and may play a role in the later steps in protein breakdown. The ATP-stimulated endoproteases appear to catalyse the rate-limiting steps in intracellular protein breakdown. However, the actual role of ATP in the degradative process is not known.

Mechanism of action of miconazole: labilization of rat liver lysosomes in vitro by miconazole.

Miconazole, a potent antifungal agent, labilizes rat liver lysosomes. Its labilizing effect is followed by measuring the release of lysosomal hydrolases, namely, acid phosphatase, beta-glucuronidase, and arylsulfatase A. The effect of miconazole is concentration dependent in the range of 10(-5) to 1.2 x 10(-4) M. However, at higher concentrations, miconazole inhibits enzyme release but does not inhibit enzyme activities per se. The effect of miconazole depends on the drug/lysosome ratio and is influenced by the pH of the incubation media, being minimal at alkaline pH. Membrane-active drugs such as nystatin, 2-phenethyl-alcohol, hexachlorophene, and digitonin have been compared with miconazole for their lysosome-labilizing action. The effect of miconazole on the lysosomal membrane is confirmed by a decrease in turbidity of the lysosomal suspension.

Studies on the mechanism of action of miconazole: effect of miconazole on respiration and cell permeability of Candida albicans.

The antifungal drug, miconazole nitrate, inhibits the growth of several species of Candida. Candida albicans, one of the pathogenic species, was totally inhibited at a concentration of approximately 10 mug/ml. Endogenous respiration was unaffected by the drug at a concentration as high as 100 mug/ml, whereas exogenous respiration was markedly sensitive and inhibited to an extent of 85%. The permeability of the cell membrane was changed as evidenced by the leakage of 260-nm absorbing materials, amino acids, proteins, and inorganic cations. The results we present clearly show that the drug alters the cellular permeability, and thus the exogenous respiration becomes sensitive to the drug.