Gas chromatographic measurement of urinary 5-fluoro-2'-deoxyuridine levels after barium salt precipitation and sephadex LH-20 cleanup

A fluorescent photoaffinity label—8-azido-1-N⁶-etheno-adenosine 5′-triphosphate (8-N₃ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F₁ATPase from Micrococcus luteus. 8-N₃ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F₁ATPase in the presence of the label and Mg²⁺ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.     

Metabolic cooperation in cell culture: studies of the mechanisms of cell interaction

Metabolic cooperation is a form of cell communication in which the mutant phenotype of enzyme deficient cells, as determined by incorporation of labeled substrates, is corrected in culture by contact with normal cells. Previous studies showed that metabolic cooperation between normal and hypoxanthine phosphoribosyl transferase deficient cells (HPRT^?) was the result of transfer of product of the enzyme, nucleotide or nucleotide derivative, from normal to mutant cells rather than transfer of enzyme or informational macromolecules leading to the synthesis of the enzyme. In the present study the nature and mechanisms involved in these cell interactions were investigated. Effective communication is observed within one hour of cell contact. Modifications of the extracellular environment including changes in osmolarity, concentration of sodium and divalent ions failed to interfere significantly with transfer. Changes of cell shape induced by cyclic nucleotides, hormones and urea also did not affect communication. Cytochalasin B which dissociates microfilaments and binds to cell membranes reduced metabolic cooperation while colcemide which dissociates microtubules had little effect. Enzymatic oxidation and iodination of cell surface structures abolished metabolic cooperation. The subcellular localization of label in donor cells is important in determining efficiency of transfer. Metabolic cooperation is efficient when radioactive label is primarily located in the nucleus and inefficient if the label is cytoplasmic. Cell lines previously classified as “non-communicating” because they lack gap junctions, ionic coupling and metabolic cooperation were shown in the present study to communicate when incubated with labeled substrates for 20 hours rather than 3. Cell communication is a more generalized phenomenon among cells in contact than previously appreciated.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation

Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of ³²P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to ³²P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low ³²P label. During 3 min treatment of the glucose starved cells with glucose the ³²P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.     

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells^1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication⁴. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2''-deoxyuridine (EdU)^5-7 with a tyramide signal amplification (TSA)⁸ protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU^5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers^9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.     

Heterogeneity of SH groups in sarcoplasmic reticulum.

The incorporation of [¹⁴C] N-ethylmaleimide reveals fast and slow-reacting sulfhydryl groups in sarcoplasmic reticulum. Two proteins react with the label: a fast-reacting glycoprotein recently isolated (Ikemoto, Cucchiaro and Garcia (1976) J. Cell Biol.$\text{70}$, 290a), and the Ca²⁺-ATPase. Labeling sarcoplasmic reticulum with a maleimide spin label gives a similar pattern. The spectra of maleimide-spin-labeled sarcoplasmic reticulum have both ‘strongly’ and ‘weakly’ immobilized components. Maleimide-spin-labeled purified Ca²⁺-ATPase, or sarcoplasmic reticulum labeled first with N-ethylmaleimide, and then with maleimide spin label, show spectra devoid of the ‘weakly’ immobilized component; the latter is enhanced in partially purified glycoprotein obtained from spin-labeled sarcoplasmic reticulum. This indicates that spectra from maleimide-spin-labeled sarcoplasmic reticulum do not reflect exclusively the state of the Ca²⁺-ATPase enzyme.     

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 µM compared to 50% protection by 405 µM 3PGA. Partial modification of the enzyme using [¹⁴C]-phenylglyoxal leads to a decrease in bothV _max,A _0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [¹⁴C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.     

Detection of conformational changes in Complex III of the respiratory chain by a maleimido spin label

Changes in the conformation of Complex III (CoQH₂-cytochromec reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochromec. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-³H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : II. RNA and its Site of Synthesis

The distribution of RNA in cells of E. coli 15 T^-U^- labeled with uridine-H³ was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H³ in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H³, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System

RNA is an essential component for the enzymic conversion of glutamate to δ-aminolevulinic acid (ALA), the universal heme and chlorophyll precursor, as carried out in plants, algae, and some bacteria. The RNA required in this process was reported to bear a close structural resemblance to tRNA^Glu(UUC), and it can be isolated by affinity chromatography directed against the UUC anticodon. Affinity-purified tRNA^Glu(UUC) from the cyanobacterium Synechocystis sp. PCC 6803 was resolved into two major subfractions by reverse-phase HPLC. Only one of these was effectively charged with glutamate in enzyme extract from Synechocystis, but both were charged in Chlorella vulgaris enzyme extract. When charged with glutamate, the two glutamyl-tRNA^Glu(UUC) species produced were equally effective in supporting both ALA formation and protein synthesis in vitro, as measured by label transfer from [³H]glutamyl-tRNA to ALA and protein. These results indicate that one of the two tRNA^Glu(UUC) species is used by Synechocystis for both protein biosynthesis and ALA formation. Both of the tRNA^Glu(UUC) subfractions from Synechocystis supported ALA formation in Chlorella enzyme extract. Escherichia coli tRNA^Glu(UUC) was charged with glutamate, but did not support ALA formation in Synechocystis enzyme extract. Unfractionated tRNA from Chlorella, pea, and E. coli, having been charged with [³H] glutamate by Chlorella enzyme extract and then re-isolated, were all able to transfer label to proteins in the Synechocystis enzyme extract.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

A Chlamydomonas reinhardii mutant with catalytically and structurally altered ribulose-5-phosphate kinase

The biochemical lesion in a light-sensitive, acetate-requiring Chlamydomonas mutant was identified. This strain, designated rpk, exhibited photosynthetic rates less than 3% of the wild-type. Analysis of photosynthetic products by high-performance liquid chromatography demonstrated an accumulation of ¹⁴C label in pentose and hexose monophosphates. After 1 min of photosynthesis in ¹⁴CO₂ these intermediates comprised 27.5% of the label in the mutant compared with 8% in the wild-type. The mutant pheno-type was caused by a 20-fold reduction in ribulose-5-phosphate (Ru5P)-kinase (EC 2.7.1.19) activity. The mutant exhibited wild-type levels of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and transketolase (EC 2.2.1.1) indicating that the mutation specifically affected Ru5P kinase. In a cross of the mutant with the wild-type, tetrad progeny segregated in a Mendelian fashion (1:1) and light-sensitivity cosegregated with reduced Ru5P-kinase activity and an acetate requirement for growth. Almost normal levels of Ru5P-kinase protein were detected in the mutant by probing nitrocellulose replicas of sodium dodecylsulfate-polyacrylamide gels with anti-Ru5P-kinase antibody. The subunit size of the mutant enzyme, 42 kDa, was identical to that of the wild-type. Isoelectric focusing of the native protein determined that the mutant protein was altered, exhibiting a more acidic isoelectric point than the wild-type protein. Thus, the molecular basis for the lesion affecting Ru5P-kinase activity in mutant rpk is a charge alteration which results in a partially impaired enzyme.Abbreviations Chl chlorophyll - Da dalton - FCCP carbonylcyanide-p-trifluorophenylhydrazone - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate     

Glucuronyl glycine,a novel N-terminus in a glycoprotein

Treatment of cutinase, an extracellular glycoprotein produced by Fusarium solani f. pisi, with NaB³H₄ at pH 7.0 generated labeled enzyme. Acid hydrolysis showed that all of the label was in an acidic carbohydrate which was identified as gulonic acid. The N-terminal amino group of the enzyme is blocked; the precursor of gulonic acid has a free reducing group and it is attached via a linkage resistant to β-elimination. Furthermore, pronase digestion of NaB³H₄-treated cutinase gave rise to a ninhydrin negative compound which contained the bulk of the ³H and this compound was identified as N-gulonyl glycine. These results strongly suggest that the amino group of glycine, the N-terminal amino acid of this enzyme, is in amide linkage with glucuronic acid.     

Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca²⁺. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca²⁺ and Mg²⁺ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.     

A direct introduction of <Superscript>18</Superscript>O isotopes into peptides and proteins for quantitative mass spectroscopy analysis

A method for direct introduction of ¹⁸O isotopes into carboxyl groups of peptides and proteins via the exchange with H₂ ¹⁸O in the presence of TFA is described. The isotope label is sufficiently stable in a wide pH range. Since the compounds labeled by this method retain their physicochemical characteristics, they can be used as an internal standard in quantitative assay of authentic compounds in the analyzed objects by means of mass spectrometry. This method is applicable to quantitative analysis of peptides and proteins in biological environments, as well as for quantitative kinetic studies of metabolism and enzyme activity. The quantitative analysis of polypeptides and proteins is combined with trypsinolysis. When necessary, the isotope label can be simultaneously introduced into all peptides and proteins in a control biosample, making it applicable as a standard for comparative analysis of experimental biosamples.     

The effects of long-chain alcohols on membrane lipids and the (Na+ + K+)-ATPase

The (Na⁺ + K⁺)-ATPase obtained from sheep kidney outer medulla is irreversibly denatured by long-chain aliphatic alcohols. The denaturation proceeds by causing a change in the structure of the membrane lipids rather than by binding directly to the protein. The alcohols decrease the ability of the membrane lipid bilayer to orient the spin label 3-(4′,4′-dimethyloxazolidinyl)-5α-androstan-17β-ol. For the low molecular weight alcohols the ability of the membrane to orient the label is completely lost while for alcohols with more than five carbons only partial loss of the orienting ability of the membrane occurs. The alcohol concentrations necessary to denature the enzyme correspond to the concentrations that produce the maximal change in the ability of the membrane to orient the label, and correlate well with the hydrophobicity of the alcohols as measured by their water-octanol partition coefficients.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

In vivo phosphorylation of enolase fromEscherichia coli

The major metabolic route for the synthesis of phosphoenolpyruvate is from 2-phosphoglycerate catalyzed by the enzyme enolase (EC 4.2.1.11). Enolase occurs at the converging point between glycolysis and gluconeogenesis and may be an important regulatory enzyme. Growth ofEscherichia coli JA 200 pLC 11-8 to stationary phase in low-phosphate medium containing³²P-orthophosphate and glucose as the carbon source resulted in incorporation of label into the enzyme. In vivo labeling of enolase was demonstrated by immunoaffinity chromatography of the labeled crude extract. In addition,³²P-enolase was identified with sodium dodecylsulfate polyacrylamide gels, two-dimensional gel electrophoresis, and Western blot analysis, followed by autoradiography.     

Persistence of embryonic RNA synthesis during facultative delayed implantation in the mouse.

The status of embryonic RNA synthesis during facultative delayed implantation in the mouse has been examined by radiolabeling in vitro and in utero, and by assay for endogenous RNA polymerase activity. Under conditions that do not activate delayed blastocysts in utero, embryos were shown to be able to transport and incorporate [³H]uridine into RNA as early as 5 min after intralumenal instillation of label on Day 5 of delay. Assay for endogenous RNA polymerase demonstrated functioning enzyme(s) in blastocysts on Day 5 of delayed implantation. Rates of incorporation of label in vitro under nonactivating conditions indicated a reduction, from normal Day 5 blastocyst levels, of 52% on Day 2 and 36% on Day 5 of delay. Relative rates of uptake of [³H]uridine by blastocysts on Day 5 of delay were reduced by approximately 60% from rates observed in predelay embryos on Day 5 of pregnancy. Estrogen-induced activation of embryos in utero was not associated with an increased relative rate of ³H]uridine uptake or incorporation during the first 24 hr following activation on Day 5 of delay. The findings demonstrate that RNA synthesis persists in the mouse blastocyst during delayed implantation, although at a somewhat reduced level. Implications of these results relevant to the maternal regulation of embryonic growth and implantation are discussed.