Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium.

Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.     

Selective incorporation of membrane proteins into proteoliposomes of different compositions.

1. Cytochrome oxidase was incorporated into preformed liposomes containing phosphatidylserine. When confronted with a mixture of liposomes, some containing phosphatidylserine and some without it, the enzyme was incorporated only into the phosphatidylserine-containing liposomes. 2. The hydrophobic proteins of the oligomycin-sensitive ATPase incubated in the presence of a mixture of liposomes with and without cytochrome oxidase were preferentially incorporated into cytochrome oxidase-containing liposomes. This selectivity was abolished by either cytochrome c or ascorbate. 3. Cytochrome oxidase incubated in the presence of a mixture of liposomes with and without the hydrophobic proteins of the ATPase was preferentially incorporated into liposomes that did not contain the hydrophobic proteins. 4. Cytochrome oxidase and the oligomycin-sensitive ATPase were preferentially incorporated into pure liposomes over bacteriorhodopsin-containing vesicles. 5. Reduced coenzyme Q (QH2)-cytochrome c reductase was incorporated randomly when incubated in the presence of a mixture of pure liposomes and liposomes containing the hydrophobic proteins of the ATPase complex. 6. The significance of the incorporation procedure as a model for membrane biogenesis is discussed.     

Incorporation of hydroxyeicosatetraenoic acids into cellular lipids of adrenal glomerulosa cells: inhibition of aldosterone release by 5-HETE

Metabolites of arachidonic acid appear to be involved in the regulation of aldosterone secretion. Adrenal cells metabolize arachidonic acid to several products including hydroxyeicosatetraenoic acids (HETEs). Since HETEs may be incorporated into the membrane lipids in some cells, we investigated whether HETEs were incorporated into lipids of adrenal glomerulosa cells and tested the influence of incorporation on aldosterone secretion. Cells were incubated with [3H] -arachidonic acid, -5-HETE, -12-HETE, -15-HETE or -LTB4. The cellular lipids were extracted and analyzed by TLC. Arachidonic acid was incorporated into all of the cell lipids with greatest accumulations in phospholipids (22%), cholesterol esters (50%), and triglycerides (21%). Uptake was maximal by 30 min. 5-HETE was incorporated into diglycerides and monoglycerides but not into phospholipids or other neutral lipids. The uptake followed a similar temporal pattern as arachidonic acid. 12-HETE was incorporated to a small extent into phospholipids, predominantly phosphatidylcholine. Neither 15-HETE or LTB4 were associated with cellular lipids. Angiotensin increased the uptake of 5-HETE and arachidonic acid into phosphatidylinositol/phosphatidylserine without altering uptake into the other lipids. When cells were pretreated with 5-HETE and washed to remove the unesterified HETE, basal aldosterone release as well as release stimulated by angiotensin, potassium and ACTH were significantly reduced. 15-HETE, which is not incorporated into cellular lipids, was without effect on aldosterone secretion. These studies indicate that 5-HETE may be incorporated into the cellular lipids of adrenal cells and may modulate steroidogenesis.     

Characterization of the stimulatory effects of PGF2alpha on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was greater than 90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (greater than 90%) was incorporated into the 2-position. PGF2alpha caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts. The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF2Alpha increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a "trap" for released arachidonic acid. The effect of PGF2Alpha was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed. The effect of PGF2Alpha depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Uptake of extracellular biotin by Escherichia coli biotin prototrophs.

Uptake of exogenous biotin by two Escherichia coli biotin prototroph strains, K-12 and Crookes, appeared to involve incorporation at a fixed number of binding sites located at the cell membrane. Incorporation was characterized as a binding process specific for biotin, not requiring energy, and stimulated by acidic pH. Constant saturation quantities of exogenous biotin were incorporated by these cells, and the amounts, which were titrated, depended on whether the cells were resting or dividing. Resting cells incorporated exogenous biotin amounting to 2% of their total intracellular biotin content. Fifty percent of the exogenous biotin was incorporated into their free biotin fraction, and 50% was incorporated into their bound biotin fraction. On the other hand, dividing cells incorporated exogenous biotin into all of their intracellular sites, 88% going into the intracellular-bound biotin fraction, and 12% going into the free biotin fraction. Calculations suggested that each cell contained approximately 3,000 binding sites for biotin. It was postulated that biotin incorporation sites might have been components of acetyl coenzyme A carboxylase located at or near the membrane.     

Biosynthetic precursors of deazaflavins.

The incorporation of 13C- and 14C-labeled precursors into 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (factor F0) was studied with growing cells of Methanobacterium thermoautotrophicum. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was incorporated into the deazaflavin and into riboflavin without dilution. Tyrosine and 4-hydroxyphenylpyruvate were incorporated into the deazaflavin and into cellular protein. 4-Hydroxybenzaldehyde was not incorporated. A reaction mechanism is proposed for the formation of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine or 4-hydroxyphenylpyruvate.     

Incorporation of phenol carbon at trace concentrations by phenol-mineralizing microorganisms in fresh water

The fate of phenol carbon at phenol concentrations ranging from 1 ng/ml to 1 microgram/ml was determined in freshwater samples. Approximately 20% of the parent phenol was incorporated into trichloroacetic acid-precipitable material by the microorganisms capable of mineralizing phenol. There was no apparent lag period before phenol incorporation commenced, and incorporation was complete within 2 h at all concentrations tested. A direct relationship was found between the initial phenol concentrations and both phenol mineralization and incorporation rates, indicating that cometabolism of phenol does not occur at concentrations that are environmentally significant. At all concentrations, approximately 80% of the initial phenol concentration was mineralized. This percentage plus the percentage of phenol incorporated at the various concentrations equaled approximately 100%. Therefore, the parent phenol does not remain in fresh water; it is either incorporated into cellular biomass or mineralized. However, the incorporated phenol carbon is subject to bioaccumulation in nature. There was no apparent lag period before thymidine was incorporated into biomass, and incorporation was complete within 4 to 8 h at all of the phenol concentrations tested. Thymidine incorporation was independent of phenol concentration at all levels tested. This is probably due to the small amount of thymidine incorporated by the phenol-mineralizing microorganisms in comparison with the amount of thymidine incorporated by the total microbial population.     

Incorporation of phenol carbon at trace concentrations by phenol-mineralizing microorganisms in fresh water.

The fate of phenol carbon at phenol concentrations ranging from 1 ng/ml to 1 microgram/ml was determined in freshwater samples. Approximately 20% of the parent phenol was incorporated into trichloroacetic acid-precipitable material by the microorganisms capable of mineralizing phenol. There was no apparent lag period before phenol incorporation commenced, and incorporation was complete within 2 h at all concentrations tested. A direct relationship was found between the initial phenol concentrations and both phenol mineralization and incorporation rates, indicating that cometabolism of phenol does not occur at concentrations that are environmentally significant. At all concentrations, approximately 80% of the initial phenol concentration was mineralized. This percentage plus the percentage of phenol incorporated at the various concentrations equaled approximately 100%. Therefore, the parent phenol does not remain in fresh water; it is either incorporated into cellular biomass or mineralized. However, the incorporated phenol carbon is subject to bioaccumulation in nature. There was no apparent lag period before thymidine was incorporated into biomass, and incorporation was complete within 4 to 8 h at all of the phenol concentrations tested. Thymidine incorporation was independent of phenol concentration at all levels tested. This is probably due to the small amount of thymidine incorporated by the phenol-mineralizing microorganisms in comparison with the amount of thymidine incorporated by the total microbial population.     

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.     

Interaction of N-ethyl-N'-nitro-n-nitrosoguanidine with nucleic acids and proteins in comparison with N-methyl-N'-nitro-N-nitrosoguanidine

Reactivity of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was studied in comparison with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The radioactivity of [guanidino-14C]-ENNG was incorporated only into the protein fraction and that of [ethyl-14C]ENNG was incorporated into DNA, RNA and protein fractions in ascites hepatoma AH7974 cells, as were those of [guanidino-14C]- and [methyl-14C]MNNG, respectively. The amounts of the binding of ENNG were less than those of MNNG, especially in the corporation of the ethyl moiety of ENNG into nucleic acid fractions. In a non-cellular system, the radioactivity of [guanidino-14C]ENNG was incorporated into proteins, preferentially into basic proteins such as cytochrome c, but was not incorporated into nucleic acids. This behavior is similar to that of [guanidino-14C]MNNG, while the amount of binding of the former was about half of that of the latter. The radioactivity of [ethyl-14C]ENNG was also incorporated into basic proteins to almost the same extent as that of [methyl-14C]MNNG. However, the binding of the ethyl moiety of ENNG to nucleic acids was much lower than that of the methyl moiety of MNNG. Horse heart cytochrome c, bovine pancreatic RNase A and regenerating rat liver chromatin had altered their biological activities to various degrees after modification by ENNG or MNNG.     

Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes

Sialoproteins isolated from the soluble fraction of rat liver could be incorporated into microsomal membranes. This incorporation was dependent on protein concentration, time, and temperature. Sodium dodecyl sulfate gel electrophoresis of membrane proteins after in vitro incorporation showed four major sugar-containing peaks and was similar to that found after in vivo labeling. Most of the incorporated protein was tightly bound to the microsomal membrane. Gel filtration and ion-exchange chromatography revealed the presence of several cytosolic glycoproteins that could be incorporated into microsomes. During prolonged centrifugation in a KBr solution with a density of 1.21 a highly labeled ([3H]glucosamine) protein (mole wt approximately to 70,000) that was actively incorporated into microsomes could be recovered in the upper region of the tube. These results demonstrate that several cytoplasmic glycoproteins of rat liver are transferred into microsomal membranes and that one of these is a lipoprotein.     

Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity.

The action of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on DNA synthesis was evaluated both in whole cells and in vitro. 9-beta-D-Arabinofuranosyl-2-fluoroadenine was converted to its 5'-triphosphate 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) in cells and then incorporated into DNA in a self-limiting manner. More than 94% of the analogue was incorporated into DNA at the 3' termini, indicating a chain termination action. In vitro DNA primer extension experiments further revealed that F-ara-ATP compared with dATP for incorporation into the A site of the extending DNA strand. The incorporation of F-ara-AMP into DNA resulted in termination of DNA strand elongation. Human DNA polymerase alpha incorporated more F-ara-AMP into DNA than polymerase epsilon (proliferating cell nuclear antigen-independent DNA polymerase delta) and was more sensitive to inhibition by F-ara-ATP. On the other hand, DNA polymerase epsilon was able to excise the incorporated F-ara-AMP from DNA in vitro. The incorporation of F-ara-AMP into DNA was linearly correlated both with inhibition of DNA synthesis and with loss of clonogenicity; thus it may be the mechanism of cytotoxicity.     

Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids

Labelled sodium isobutyrate [(CD3)2-CHCOONa] was added to the culture medium of Streptomyces fradiae and up to 14 atoms of deuterium were found to be incorporated into a molecule of tylosin aglycone (tylactone). This observation is in accordance with the data in the literature. When fatty acids were analyzed, as much as 34% of the isobutyrate incorporated into the cell was formed to be transformed into butyrate that was used for the synthesis of even, straight-chain fatty acids; 57% of the labelled isobutyrate was incorporated into the even isoacids, whereas 9% was degraded to propionate and further used for the synthesis of the odd acids.     

Cholesterol incorporation into bacterial membranes.

The wall-covered bacteria Micrococcus lysodeikticus, Bacillus megaterium, and Proteus mirabilis incorporated exogenous cholesterol into their cytoplasmic membrane in quantities resembling those incorporated by sterol-nonrequiring mycoplasmas. Cholesterol incorporation into the outer membrane of P. mirabilis was much more restricted than into the cytoplasmic membrane.     

Reconstruction of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Using the freezing-thawing procedure, a highly purified preparation of PPase from R. rubrum chromatophore membranes was incorporated into soybean phospholipid liposomes. The activity of reconstituted PPase was increased in the presence of the uncoupler, FCCP, and the antibiotics, valinomycin (+KCl) and nigericin (+KCl). Oligomycin did not exert any inhibiting action, while imidodiphosphate and NaF significantly decreased the activity of the PPase incorporated into the liposomes. Preincubation of both PPase and ATPase prior to their incorporation into the liposomes did not affect the activity of the reconstituted enzyme. It was concluded that the PPase from R. rubrum chromatophores when incorporated into the liposomes may function as a proton pump independently of the ATPase.     

Incorporation of [3H]glucosamine into keratin-related polypeptides in pig epidermis

Metabolic labelling studies have provided evidence for glycosylated keratins in cultured pig epidermis. [3H]Glucosamine was incorporated into five major particulate polypeptides of Mr 68 000, 61 000, 57 000, 53,000 and 48,000. Radioactivity was present in protein-bound carbohydrate. Non-enzymic glycation was excluded. Labelling was largely unaffected by tunicamycin indicating that radioactivity was incorporated mainly into O-linked oligosaccharides. These [3H]glucosamine-labelled components were closely related to keratins since they had a similar electrophoretic mobility to polypeptides of purified pig prekeratin, they were immunoprecipitated by anti-prekeratin serum and they were incorporated into reconstituted, intermediate-sized, keratin filaments.     

Altered specificity of transfer-ribonucleic acid nucleotidyltransferase in the presence of manganese

1. s-RNA nucleotidyltransferase incorporated CMP into phosphodiesterase-treated s-RNA more rapidly in the presence of Mg(2+) (10mm) than in the presence of Mn(2+) (2mm). UMP was incorporated more rapidly in the presence of Mn(2+), and at high ionic strength the incorporation of CMP was also more rapid in the presence of Mn(2+). 2. The capacity of phosphodiesterase-treated s-RNA for CMP, UMP and AMP was increased in the presence of Mn(2+). Terminal sequences of more than one UMP or AMP residue were synthesized, but these atypical reactions were inhibited when CTP was added. CMP was incorporated rapidly to form -pCpC terminal sequences and then more slowly as longer chains were formed, but very little CMP was incorporated into s-RNA-pCpCpA. 3. CMP was incorporated into phosphodiesterase-treated 5s RNA and ribosomal RNA to form short chains of polyC attached to the primer RNA. This reaction was inhibited by the presence of s-RNA. 4. A small Mn(2+)-dependent incorporation of CMP was also primed by poly(A).(U) and poly(C).(I).     

Phosphorylation and degradation of exogenous phosphatidylinositol incorporated into Friend erythroleukemic cells

Phosphatidyl[2-3H]inositol (PtdIns) obtained from rat skeletal muscle and yeast was introduced into Friend erythroleukemic cells by use of the PtdInstransfer protein or by spontaneous route. The mammalian PtdIns incorporated by the transfer protein appeared metabolically inert while the spontaneously incorporated PtdIns was both phosphorylated to PtdIns-4-phosphate (i.e. 30% of the total PtdIns incorporated) and converted into lyso-PtdIns (i.e. 20% of the total PtdIns incorporated); formation of PtdIns, 4,5-bisphosphate was minimal. The extensive metabolism of the spontaneously incorporated PtdIns strongly suggests that this PtdIns does not rapidly equilibrate with the endogenous PtdIns pools. A similar spontaneous incorporation of yeast PtdIns was accompanied by a negligible degree of phosphorylation and hydrolysis. Evidence is provided that this difference in metabolism reflects the absence of arachidonate in the yeast PtdIns.     

Incorporation of Mevalonic Acid into a Lipid Intermediate in Cell Wall Synthesis and Nucleic Acid Fraction by Lactobacillus heterohiochii

It was, using the particulate enzyme from Micrococcus lysodeikticus, revealed that most of 2-¹⁴C-mevalonic acid incorporated into the cell of Lactobacillus heterohiochii H-1 was incorporated into the lipid intermediate of cell wall biosynthesis. About 10% of the radioactivities incorporated into the cells was, however, found in nucleic acid fraction which was extracted from lysozyme treated cells with phenol. Most of the radioactivities in the nucleic acid fraction was eluted at the beginning of the elution pattern from Sephadex G-200 or MAK-column. The material is different from tRNA and rRNA.