A new purification procedure for Clostridium difficile enterotoxin

$\text{Clostridium difficile}$ produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A₁ = 41,500, A₂ = 16,000) and its pI is 3.5.     

Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clostridiumperfringens

Rat liver parenchymal cells (hepatocytes) were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium in collagen-coated culture dishes. Glucagon, in combination with dexamethasone, induced α-aminoisobutyric acid transport in these cells. Addition of purified $\text{Clostridium}\text{perfringens}$ enterotoxin to hepatocytes preinduced by glucagon and dexamethasone rapidly depressed (but did not abolish) α-aminoisobutyric acid transport. The toxin effect was dose dependent: 1000 or 300 ng/ml produced maximal depression whereas 100 or 40 ng/ml were without effect in 120 minutes. The effect was eliminated by pretreating the toxin with heat or specific antisera. The effect of enterotoxin on α-aminoisobutyric acid transport in two cultured rat hepatoma cell lines (H4-II-E-C3 and McA-RH 7777) was also investigated. Only the McA-RH 7777 cells were sensitive to the toxin suggesting that the enterotoxin may interact with specific membrane components of normal rat liver cells which are also present on some (but not all) cancerous rat liver cells.     

Structure of a ribosomal 5S RNA from a mushroom, Coprinus cinereus

The nucleotide sequence of the 5S rRNA from a mushroom, $\text{Coprinus cinereus}$, was determined to be: pAUCCACGGCCAUACGACUCUGAAAGCACCGCACCGCAUCCCGUCCGAUCUGCGCAGUUAACCAGAGUGCCGCUCAGUUAGUACCACGGUGGGGGACCACGCGGGAAUCCUGGGUGCUGUGGUU. This sequence is consistent with current models for the secondary structure of 5S RNAs and indicates a very high degree of sequence conservation among the most highly evolved fungi. Sequence heterogeneity was not evident in this fungus suggesting that the more highly evolved fungi may not contain the dispersed pattern of 5S rRNA genes which have been observed in intermediate fungi such as $\text{Neurospora}$ (Selker, E.U., and Yanofsky, C. (1981) Cell $\text{24}$, 819–828.)     

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.     

Studies on the red oxidase (cytochrome o) of Azotobacter vinelandii

In an attempt to isolate and to study the electron transport system of $\text{Azotobacter vinelandii}$, we have isolated and purified a membrane-bound cytochrome $\text{o}$. The cytochrome $\text{o}$, purified as a detergent (Triton X-100) and hemoprotein complex, contained 1.6 nmoles heme per mg of protein. Cold-temperature spectrum showed that no other cytochrome was associated with the purified preparation, and electrophoresis revealed that only one type of hemoprotein was obtained. The purified cytochrome $\text{o}$ reacted with both carbon monoxide and cyanide readily. Only in the reduced form did it combine with carbon monoxide, whereas the oxidized form reacted with cyanide. An “oxygenated” form of the cytochrome $\text{o}$ was demonstrated to be spectrally distinguishable from both the oxidized and the reduced forms.     

Structure of rhodotorucine A, a novel lipopeptide,inducing mating tube formation in Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ is a peptidyl factor which induces mating tube formation in $\text{Rhodosporidium}\text{toruloides}$. The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine $\text{A}$ was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine $\text{A}$. H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH     

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.     

Release and purification of micrococcus lysodeikticus ATPase from membrane extracted with n-butanol

The ATPase associated with the membranes of Micrococcus ysodeikticus has been released into the aqueous phase (i.e. solubilized) by extracting the membranes with $\text{n-}\text{butanol}$ in a two-phase system modified from the procedure of Maddy, A.H. (164) Biochim. Biophys. Acta 88, 448–449. A procedure for the release and purification of the ATPase from the membranes extracted with $\text{n-}\text{butanol}$ is described as an alternate method to that previously used for the shock-wash ATPase. Upon extracting the membrane suspensions with $\text{n-}\text{butanol}$ the soluble ATPase released into the buffer phase no longer exhibits stimulation by trypsin in contrast to the shock-wash type of ATPase. As shown by Salton, M. R. J. and Schor, M. T. (1972) Biochem. Biophys. Res. Commun. 49, 350–357, the shock-wash ATPase possesses associated protein(s) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis whereas these are absent from the purified ATPase released by the $\text{n-}\text{butanol}$ method. The specific activities of the purified ATPase released by the two methods were generally similar, the $\text{n-}\text{butanol}$ type being consistently somewhat higher.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

Effects of adrenal steroid activator protein on the conversion of various 20- and 22-hydroxycholesterols to pregnenolone by adrenal mitochondrial enzymes

A heat-stable protein activator from bovine adrenal cortex mitochondria stimulates the conversion of cholesterol to pregnenolone in crude extracts of adrenal mitochondria, and resembles in some of its properties, the sterol carrier protein of liver (Kan $\text{et}\text{al}$. $\text{Biochem}$. $\text{Biophys}$. $\text{Res}$. $\text{Commun}$. $\text{48}$, 423–429, 1972). We have shown that activator preparations also stimulate highly purified adrenal enzyme preparations comprising four components: cytochrome P-450 specific for side chain cleavage, adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Furthermore, this activator stimulates the conversion not only of cholesterol, but also of (20S)-20-hydroxycholesterol, (22$\text{R}$)-22-hydroxycholesterol, and (20$\text{R}$, 22$\text{R}$)-20,22-dihydroxycholesterol to pregnenolone. Our findings provide additional evidence that the steroid-activator complexes are the substrates for the side chain cleavage enzyme and that the monohydroxy and dihydroxycholesterols are true intermediates in the conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria.     

Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells.

Cells resistant to $\text{Clostridium perfringens}$ enterotoxin were selected from cultures of highly sensitive Vero (African green monkey kidney) cells. Studies were done with the sensitive and resistant cells to determine the relationship between binding and biological activity. Binding studies using ¹²⁵I-enterotoxin revealed the apparent existence of high and low affinity binding sites for the enterotoxin on both cell types. The binding site density on resistant cells was found to be $\text{1}\text{10}$ that of sensitive cells. It was found that, even with high doses of enterotoxin, only partial affect upon DNA synthesis, membrane permeability, and plating efficiency was noted in resistant cells. It is concluded that without specific binding there is little or no ability of the enterotoxin to effect biological activity in cells.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

The amino acid sequence of a 27-residue peptide released from the alpha-chain carboxy-terminus during the plasmic digestion of human fibrinogen.

The amino acid sequence of a 27-residue peptide released during the early stages of the plasmin digestion of human fibrinogen has been determined. The corresponding cyanogen bromide fragment has also been isolated from the purified α-chains of fibrinogen, although a separable fraction of those chains lack the fragment, evidently because of $\text{in}$$\text{vivo}$ degradation. The peptide is the carboxy-terminal segment of native α-chains.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Purification and characterization of a heat-stable enterotoxin of Vibrio mimicus

A heat-stable enterotoxin produced by Vibrio mimicus (VM-ST) was studied. VM-ST was purified from a culture supernatant of V. mimicus strain AQ-0915 by ammonium sulfate fractionation, hydroxyapatite treatment, ethanol extraction, column chromatography on both SP-Sephadex C-50 and DEAE-Sephadex A-25, and HPLC, and the recovery rate was about 15%. Purified VM-ST was heat-stable. VM-ST activity was cross-neutralized by anti-STh antiserum. The amino acid composition of the purified VM-ST was determined 17 amino acid residues in the following sequence: Ile-Asp-Cys-Cys-Glu-Ile-Cys-Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn. This composition and sequence were identical to those of V. cholerae non-O1-ST. These results clearly demonstrate the production of a characteristic VM-ST by V. mimicus.     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

A new site-specific endonuclease from Streptomyces lavendulae (SlaI)

A new restriction-like endonuclease, SlaI, was found and partially purified from Streptomyces lavendulae ATCC8664. This endonuclease cleaved bacteriophage lambda DNA at only one site, and cytosine-substituted bacteriophage T4 DNA at 16 sites. The recognition sequence was determined by using SlaI fragments of cytosine-substituted bacteriophage T4 DNA. The hexanucleotide recognized by SlaI endonuclease was

$\text{5′-}\text{C}\text{?}\text{T}\text{-}\text{C}\text{-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-3′}$

$\text{3′-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-}\text{C}\text{-}\text{A}\text{-↑}\text{C}\text{-5′}$

with the sites of cleavage as indicated by the arrows. Therefore, SlaI endonuclease was an isochizomer of XhoI endonuclease.     

Triton X nonionic surfactants reversibly inhibit the EDTA/KC1 ATPase activity of myosin

The nonionic octylphenoxy polyethoxy series of surfactants, Triton X, reversibly inhibited the $\text{EDTA}\text{KCl}$ ATPase activity of purified rabbit skeletal muscle myosin at concentrations at or below their reported critical micelle concentrations. The maximum degree of enzyme inhibition increased with ethoxy content to 88% (with Triton X 102 average ethoxy content, 12.5 per molecule). The results suggest that binding of the surfactant to the myosin molecule occurs below the critical micelle concentrations and that the hydrophilic ethoxy chain forms a diffusion barrier against approach of ATP to the enzyme's active site. This model has implications for the organization of myosin in the plasma membrane of phagocytes.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.