Mammalian tRNA sulfurtransferase: properties of the enzyme in rat liver.

Transfer RNA sulfurtransferase activity was detected in 105,000 x g supernatant preparations from rat liver and several other rat tissues. Sulfur is transferred from [35S] cysteine to tRNA in a reaction which also requires ATP, Mg2+, and supernatant protein. While [35S] beta-mercaptopyruvate appeared to be a substrate for this enzyme, the reaction product was sensitive to deacylation and the reaction was inhibited by [32S] cysteine. Of the various nucleic acids tested, only tRNAs were effective sulfur acceptors, with rat liver tRNA being the poorest substrate. The [35S] reaction product was sensitive to ribonuclease, cochromatographed with tRNA on methylated-albumin kieselguhr columns, and was converted to nucleotide material after alkaline hydrolysis. DEAE-cellulose chromatography of the neutralized [35S] nucleotide digest revealed a single thionucleotide peak. These studies demonstrate that tRNA sulfurtransferase is present in various rat tissues, and that the requirements of the liver enzyme are similar to those of bacterial enzymes.     

Methicillin resistance in Staphylococcus epidermidis. Relationship between the additional penicillin-binding protein and an attachment transpeptidase

The penicillin-binding proteins (PBP) of a methicillin-resistant strain of Staphylococcus epidermidis, 100,604 p+m+ and a non-isogenic sensitive strain, p-m- were characterised. The presence of a novel PBP, produced by the methicillin-resistant strain of S. epidermidis, with an Mr identical to that of PBP2' in Staphylococcus aureus 13,136 p-m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30 degrees C, in media containing beta-lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non-growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100,604 p+m+ and from PBP2' in S. aureus 13,136 p+m+ lends support to the theory that the additional DNA encoding PBP2' in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice-versa, indicated that there was a strong correlation between the presence of PBP2' and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of beta-lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of beta-lactam antibiotics at either 40 degrees C or 30 degrees C suggests that there is little relationship between the production of this enzyme and of PBP2' other than the fact that beta-lactam antibiotics are common inducers of both.     

Characterization of an RNA-dependent RNA polymerase activity associated with measles virus

An RNA-dependent RNA polymerase activity has been found copurifying with measles virus infectivity and complement-fixing antigen in three Vero cell-grown variants of measles virus: the attenuated Edmonston B strain, the natural non-attenuated Edmonston strain, and a subacute sclerosing panencephalitis isolate, IP-3. Incubation of purified measles virions with immunoglobulin G derived from sera of monkeys hyperimmunized against measles specifically removes activity sedimenting in the density region of measles virions. The requirements of the reaction, which is RNase sensitive, are similar to those reported for other paramyxovirus-associated activities, including detergent, divalent cation, ribonucleoside triphosphates, and a reducing agent. The size classes of RNA synthesized correspond to those found in measles-infected cells, including 50, 35, and 16 to 20S. The product RNA of the Edmonston B virus-stimulated reaction was rendered RNase resistant by annealing with RNA extracted from purified Edmonston B virions. RNA from uninfected Vero cells was ineffective in the annealing reaction.     

Transfer ribonucleic acid nucleotidyltransferase activity in virions of type-C viruses.

A nucleotidyltransferase activity has been found associated with a number of mammalian and avian oncornaviruses. This activity catalyzes the incorporation of adenosine monophosphate and cytosine monophosphate into acid insoluble forms. The transferase activity from Rauscher murine leukemia virus has been characterized. The endogenous reaction is stimulated by various tRNAs particularly the 4S RNA isolated from Rauscher leukemia virus, whereas other RNAs have no effect. The product of the reaction is alkali and RNase sensitive, insensitive to DNase, and its size is similar to tRNA. Finally, the terminal nucleotide analysis of the product of the reaction indicates the presence of a CCA terminus. The properties of the activity found in the type-C viruses are in accord with those of known tRNA nucleotidyltransferases from other sources.     

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry

The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3'-diaminobenzidine (DAB). In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane. In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.     

Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation.

Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.     

Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro.

The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.     

Initiation of lambda DNA replication in vitro promoted by isolated P-gene product.

The product of the P gene of bacteriophage lambda was isolated from heat-induced lambda-lysogenic Escherichia coli cells. It was found to bind to DNA, to be devoid of nuclease activity acting on double-stranded lambda DNA and of nicking/closing activity. Initiation of lambda DNA replication promoted by the P-gene product in a complementation assay in vitro was sensitive to rifampicin. Sedimentation analysis of the products and their hybridization to separated lambda DNA strands indicate that lambda DNA was formed in a reaction similar to ring-to-ring replication in vivo. The reaction was symmetric from the beginning, i.e. both lambda DNA strands were copied without delay.     

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.     

Formation of G-quadruplex-hemin DNAzyme based on human telomere elongation and its application in telomerase activity detection

In the present study, a chemiluminescence method for sensitive detection of human telomerase activity was developed based on the formation of G-quadruplex-hemin DNAzyme. In the presence of telomerase, the telomerase substrate (TS) primer elongated and a long single-strand DNA containing the telomere repeat units (TTAGGG)n was formed. When K(+) was introduced, the telomere repeat units could form G-quadruplex and then combined with hemin to form DNAzymes which could stimulate the generation of chemiluminescence (CL) in the presence of luminol and H(2)O(2). The amount of telomerase elongation product was controlled by the content of telomerase extracted from HeLa cells, so the amount of DNAzymes and the intensity of chemiluminescence signal were all related to the number of HeLa cells. Using this simple method, the telomerase activity extracted from 100 cultured cancer cells could be detected without the polymerase chain reaction (PCR) amplification of telomerase elongated product.     

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.     

Ultrastructural demonstration of endogenous peroxidatic activity in mammalian arterial wall

Summary Endogenous peroxidatic activity has been demonstrated at the ultrastructural level in large arteries of rabbit and rat using diaminobenzidine. The reaction was positive in endothelial cells of both species and also in the smooth muscle cells of rat arteries. The reaction product was localized in the nuclear envelope and endoplasmic reticulum of the reactive cells. Since the enzymatic activity was extremely sensitive to fixation, best visualization was obtained in unfixed, directly incubated tissues in which additional mitochondrial staining occurred due to the activity of endogenous cytochrome c/cytochrome oxidase system. The peroxidatic activity was partially sensitive to cyanide and could be completely abolished by azide and aminotriazole. It has been suggested that the observed endogenous peroxidatic activity of the arterial wall components reflects the activity of prostaglandin endoperoxide synthetase and, indirectly, production of prostacyclin (PGI₂).Preliminary results were presented at the VIth International Histochemistry and Cytochemistry Congress in Brighton, England, 17–22 August 1980     

Ultrastructural cytochemical studies of plasma membrane phosphatase activities during the HeLa S3 cell cycle.

Alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg2+-activated ATPase (Mg-ATPase) and Ca2+-activated ATPase (Ca-ATPase) were studied in sychronized HeLa S3 cells with cytochemical methods and electron microscopy. It was found that AP activity, as determined by the deposition of lead phosphate reaction product (r.p.) was most active in mitotic (M), early and middle G1 cells, less active in late G1 and almost undetectable in S phase cells. Most AP enzyme activity was found to be associated with undulations (mainly microvilli) of the plasma membrane. Fluctuations and the redistribution of 5'N were also observed; the reaction for 5'N was positive in all phases of the cell cycle studied, it was strongest in M cells and in the majority of middle G1 cells. Mg-ATPase activity was present in the plasma membranes of cells throughout the cell cycle, but did not show noticeable fluctuations in activity and distribution. Ca-ATPase activity appeared in plasma membranes and in limited areas of cell nuclei but was evident only in S phase cells. The results of the present study confirm and extend previous biochemical observations and indicate that changes in membrane phosphate activities are associated with enzyme activity redistributions within the plasma membrane during the HeLa S3 cell cycle.     

Oxidation of trans- and cis-1,2-cyclohexanediol by Gluconobacter oxydans

Summary The enzymatic oxidation of 1,2-cyclohexanediol and related substrates by Gluconobacter oxydans (ATCC 621) was investigated. At low pH, membrane-bound enzymes were active and at high pH, NAD-dependent, soluble enzymes showed activity. Whole bacterial cells were used to catalyze some bioconversions. Racemic trans-1,2-cyclohexanediol was oxidized at pH 3.5 to give (R)-2-hydroxycyclohexanone (96% e.e.) and at pH 8.0 the same substrate was oxidized to (S)-2-hydroxycyclohexanone (97% e.e.). The latter conversion was severely inhibited by the reaction product while the former was not significantly product inhibited. (S)-2-hydroxycyclohexanone (97% e.e.) was also prepared from cis-1,2-cyclohexanediol by oxidation with G. oxydans cells at pH 3.5 in a reaction which continued to 100% conversion.     

Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37 degrees C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy- N-acetylneuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.     

RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.     

Histochemistry of hydrolytic enzymes in resting submandibular glands of rabbits

Summary The distribution of several hydrolytic enzymes was investigated in rabbit submandibular glands at both the light and electron microscopical levels. Glands were fixed by either immersion or perfusion fixation with a variety of fixatives containing 1–2% glutaraldehyde and 2–4% formaldehyde in 0.1m cacodylate buffer at pH 7.2. Light microscopically, the acinar cells showed some staining for ATPase, acid phosphatase and nonspecific esterases but showed weak staining for thiamine pyrophosphatase. Acid phosphatase staining occurred most strongly in granular tubule cells. Staining for esteroproteases was confined to the periluminal rims of intercalary and striated ducts. Alkaline phosphatase was very sensitive to glutaraldehyde and was confined to myoepithelial cells.Electron microscopical observations revealed the presence of acid phosphatase reaction product in lysosomes, immature granules and in GERL-like structures, the last being much more conspicuous in the granular tubule cells. ATPase reaction product was localized to the basal and luminal plasma membranes and lumina of both acinar and granular tubule cells. The Golgi complex of these two types of cells exhibited only moderate amounts of reaction product for thiamine pyrophosphatase. Alkaline phosphatase activity, on the other hand, was exclusively localized to myoepithelial cells in their plasma membranes and sometimes in the nuclear envelope.     

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.     

Enhanced light microscopic visualization of oxidase activity with the cerium capture method

Visualization methods for the light microscopic detection of the activity of oxidases after being localized with cerium ions as reported by Angermüller and Fahimi (1988a, b) are not suitable for the demonstration of H2O2-genrating oxidases at sites with low activity. Therefore, the cerium-diaminobenzidine (DAB) visualization procedure of these authors was modified. Nickel or cobalt ions were added to the DAB solution together with small amounts of H2O2. Visualization was performed in a one-step-method. This modified visualization technique enables light microscopic detection of amino acid oxidase activity in kidney and liver cells where it was found with the original method but the amounts of final reaction product were considerably higher. Moreover, the DAB-nickel-H2O2 and DAB-cobalt-H2O2 procedures were more sensitive than the cerium-lead method of Angermüller and Fahimi (1988a, b). The method appeared to be specific, because final reaction product was not found after control incubation. Especially the DAB-nickel-H2O2 procedure can also be used for immunohistochemistry when glucose oxidase serves as the enzyme label.     

Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.