Two type I restriction enzymes from Salmonella species. Purification and DNA recognition sequences

We have purified the type I restriction enzymes SB and SP from Salmonella typhimurium and S. potsdam, respectively, and determined the DNA sequences that they recognize. These sequences resemble those previously determined for the type I enzymes, EcoB, EcoK and EcoA, in that the specific part of the sequence is divided into two domains by a spacer of non-specific sequence that has a fixed length for each enzyme. Two main differences from the previously determined sequences are seen. Both of the new sequences are degenerate and one of them, SB, has one trinucleotide and one pentanucleotide-specific domain rather than the trinucleotide and tetranucleotide domains seen for all of the other enzymes. The only conserved features of the recognition sequences are the adenosyl residues that are methylated in the modification reaction. For all of the enzymes these are situated ten or 11 base-pairs apart, one on each strand of the DNA. This suggests that the enzymes bind to DNA along one face of the double helix making protein-DNA interaction in two successive major grooves with most of the non-specific spacer sequence in the intervening minor groove.     

Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis

A synthetic medium was developed in which the presence of phenylalanine ammonialyase (PAL) in the yeast Rhodotorula glutinis was dependent on the addition of l-phenylalanine. The appearance of PAL activity occurred during mid- to late log phase regardless of the time of l-phenylalanine introduction into the medium. Maximum levels of PAL activity were followed by a rapid decline in both total and specific activity. These changes were accompanied by comparable fluctuations in PAL antigen levels as measured by rocket immunoelectrophoresis. Proteins of yeast grown in the presences of l-phenylalanine were radiolabeled in vivo with l-[³H]leucine. The labeled protein was immunoprecipitated with anti-PAL serum and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A labeled protein comigrated with subunits of authentic PAL. These data support the hypothesis that de novo synthesis of PAL in R. glutinis is l-phenylalanine dependent.     

Starch phosphorylase from tapioca leaves: Absence of pyridoxal phosphate

Starch phosphorylase from tapioca leaves has been purified to homogeneity, using the technique of ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, filtration through Sephadex G-100 and Sephadex G-200, and DEAE-Sephadex chromatography. The enzyme has a molecular weight of 450,000, as determined by gel filtration through Sephadex G-200 and contains 22 sulfhydryl groups per mole of the enzyme protein. Several types of evidence indicate the absence of pyridoxal 5′-phosphate as a prosthetic group of the enzyme. The kinetic data show a sequential type of the reaction mechanism. The enzyme activity is inhibited by tyrosine (K_i = 2.15 mm).     

Crystallographic data for haemoglobin from the lanceolate fluke Dicrocoelium dendriticum

Two crystal modifications of the monomeric haemoglobin from the flatworm Dicrocoelium dendriticum have been obtained by vapour diffusion against buffered polyethylene glycol solutions. Both the triclinic and hexagonal crystals contain cyanomethaemoglobin. The triclinic modification, space group PI, $\text{a = 37.1}\text{A}\text{?}\text{, b = 39.9}\text{A}\text{?}\text{, c = 49.0}\text{A}\text{?}\text{, α = 88.8}\text{°}\text{, β = 76.8}\text{°}\text{, γ = 64.6}\text{°}$, with two molecules, M_r = 16,750 each, per unit cell, has been selected for a detailed crystallographic study.     

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase high-pressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.     

Immunoassay for bovine serum thymopoietin: discrimination from splenin by monoclonal antibodies

A polyclonal rabbit anti-bovine thymopoietin antiserum was used to develop a radioimmunoassay and sandwich enzyme-linked immunosorbent (ELISA) assay for the thymic hormone thymopoietin. Both assays showed slightly less sensitivity for the closely related splenic hormone splenin (SP) than thymopoietin (TP) and markedly less sensitivity for the human as compared with the bovine polypeptides. A number of murine monoclonal antibodies specific for bovine thymopoietin were generated; they were unreactive with bovine splenin and were also unreactive with human thymopoietin and splenin. A sandwich ELISA using these monoclonal anti-TP antibodies together with polyclonal rabbit anti-TP was specific for bovine thymopoietin and measured 300-500 ng/ml thymopoietin in bovine serum. Similar approaches are being pursued to develop an immunoassay for thymopoietin in human serum.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.     

A prolyl endopeptidase from murine macrophages, its assay and specific inactivation

The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.     

Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.     

Hydride transfer from transition metal hydrides to dihapto acyl ligands

The dihapto acyl ligand in Cp₂Zr[C(O)R]X, (R = Me, Ph; X = Me, Cl) is subject to hydrogen transfer from Cp₂MH₂ (M = Mo, W), Cp₂ReH and CpReH₄(PMe₂Ph). The initial products are bimetallic dimers of the type Cp₂XZrOCH(R)ML_n. The fate of this bimetallic species is highly dependent upon the Group VIB metal when Cp₂MH₂ is the hydride source. For M = Mo, a second hydrogen migrates to the acyl carbon, yielding Cp₂Zr(OEt)Me and products derived from Cp₂Mo. For M = W, CO bond scission occurs with retention of the WC bond, to yield the carbene complexes Cp₂WC(C)R along with various oxyzirconium products. Filled d orbitals are not necessary on the hydride source; Cp₂NbH₃ also readily reduces the acyl in Cp₂Zr[C(O)Me]Me.     

Proteoglycans from the substratum adhesion sites of MSV-transformed Balb/c 3T3 cells

Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 m guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material—high-density G_A1 which contained some HA but principally CS and HS; intermediate-density G_A2 which contained only HA; and low-density G_A3 which contained some HA and principally glycoprotein. R-SAM gradients contained no G_A2 but a sizable amount of “low-density” HA in G_A3. When centrifuged under dissociative conditions, most of G_A1 and all of G_A2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have “matured”, indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.     

Bile acids XLII. Preparation of 5-alpha-cholestan-26-oic acids from kryptogenin

3β, 7α, 26-Triacetoxy-5α-cholestane was prepared from 25R-3β, 26-diacetoxy-5α-cholestan-7α-ol, and partially hydrolyzed with potassium carbonate in methanol-benzene. The three acetylated products thus obtained were characterized by thin layer and gas liquid chromatography, and mass spectrometry. By oxidation and alkaline hydrolysis, 3β, 7α-diacetoxy-5α-cholestan-26-ol was converted to 3β, 7α-dihydroxy-5α-cholestanoic acid. 7α, 26-Diacetoxy-5α-cholestan-3β-ol was characterized as indicated. The third product, 7α-acetoxy-5α-cholestane-3β, 26-diol was oxidized to 3-oxo-7α-acetoxy-5α-cholestanoic acid which was reduced catalytically and hydrolyzed to provide 3α, 7α-dihydroxy-5α-cholestanoic acids and its 3β-isomer. By comparison of the specific rotation of this sample of 3α, 7α-dihydroxy-5α-cholestanoic acid derived from 25R-kryptogenin with a similar product derived from arihydro-5α-cyprinol obtained from carp bile, the latter derivative appears to be primarily the 25S material.     

Purification and characterization of intact cytochrome b5 from yeast microsomes

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.     

Self cleavage of a precursor RNA from bacteriophage T4

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Leakage of sucrose from phosphatidylcholine liposomes induced by interaction with serum albumin

Liposomes composed of rat-liver phosphatidylcholine rapidly lose entrapped sucrose when incubated in presence of blood or of solutions of bovine serum albumin. The phenomenon can not be ascribed to phospholipase A activity, since no such activity towards phosphatidylcholine substrates could be detected in various albumin preparations. Upon gel filtration on Sepharose 4B or Sephadex G-100 of incubated mixtures of radioactive liposomes and albumin, association of phosphatidylcholine with the albumin could be demonstrated. No measurable quantities of protein were found associated with liposomes. The albumin-associated phosphatidylcholine is hydrolyzed by pancreatic phospholipase A more slowly than free liposomal phosphatidylcholine, indicating a non-lamellar orientation of the associated phospholipid. The binding of phosphatidylcholine to albumin proceeds at a slow rate: increase of the amount of phosphatidylcholine bound continues over a period of several hours reaching a maximum at approx. 1 mol of phosphatidylcholine per mol of albumin. The process is reversible as indicated by transfer of albumin-associated radioactive phosphatidylcholine to unlabeled liposomes. The association between albumin and phosphatidylcholine is believed to be of the same type as described recently by Jonas) Jonas, A. (1976) Biochim. Biophys. Acta 427, 325–336)). The consequences of these observations are discussed with respect to the use of liposomes as carriers to introduce substance into cells.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.